Nucleic acids for inhibiting expression of LPA in a cell

ABSTRACT

The present invention relates to products and compositions and their uses. In particular the invention relates to nucleic acid products that interfere with the LPA gene expression or inhibit its expression, preferably for use as treatment, prevention or reduction of risk of suffering cardiovascular disease such as coronary heart disease or aortic stenosis or stroke or any other disorder, pathology or syndrome linked to elevated levels of Lp(a) particles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/293,303, filed May 12, 2021, which is a 35 U.S.C. § 371 nationalphase entry of International Application No. PCT/EP2019/081158, filedNov. 13, 2019, which claims priority to International Patent ApplicationNo. PCT/EP2018/081106, filed Nov. 13, 2018, and European PatentApplication No. 19174466.3, filed May 14, 2019. The disclosures of eachapplication are hereby incorporated by reference in their entireties.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

A Sequence Listing, which is a part of the present disclosure, issubmitted concurrently with the specification as a text file. The nameof the text file containing the Sequence Listing is“56744A_Seqlisting.txt.” The Sequence Listing was created on Dec. 21,2021, and is 38,552 bytes in size. The subject matter of the SequenceListing is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to products and compositions and theiruses. In particular the invention relates to nucleic acid products thatinterfere with the LPA gene expression or inhibit its expression. Suchtherapeutic Lp(a) lowering therapy serves to prevent and reduce the riskof suffering stroke, atherosclerosis, thrombosis and cardiovasculardiseases such as coronary heart disease and aortic stenosis or any otherdisorder, pathology or syndrome linked to elevated levels of Lp(a)particles.

BACKGROUND

Double stranded RNAs (dsRNA) able to complementarily bind expressed mRNAhave been shown to be able to block gene expression (Fire et al., 1998,Nature. 1998 Feb. 19; 391(6669):806-11 and Elbashir et al., 2001,Nature. 2001 May 24; 411(6836):494-8) by a mechanism that has beentermed RNA interference (RNAi). Short dsRNAs direct gene specific, posttranscriptional silencing in many organisms, including vertebrates, andhave become a useful tool for studying gene function. RNAi is mediatedby the RNA induced silencing complex (RISC), a sequence specific, multicomponent nuclease that degrades messenger RNAs homologous to thesilencing trigger loaded into the RISC complex. Interfering RNA (termedherein iRNA) such as siRNAs, antisense RNAs, and micro RNAs areoligonucleotides that prevent the formation of proteins by genesilencing i.e. inhibiting gene translation of the protein throughdegradation of mRNA molecules. Gene silencing agents are becomingincreasingly important for therapeutic applications in medicine.

According to Watts and Corey in the Journal of Pathology (2012; Vol 226,p 365 379) there are algorithms that can be used to design nucleic acidsilencing triggers, but all of these have severe limitations. It maytake various experimental methods to identify potent iRNAs, asalgorithms do not take into account factors such as tertiary structureof the target mRNA or the involvement of RNA binding proteins.Therefore, the discovery of a potent nucleic acid silencing trigger withminimal off target effects is a complex process. For the pharmaceuticaldevelopment of these highly charged molecules it is necessary that theycan be synthesised economically, distributed to target tissues, entercells and function within acceptable limits of toxicity.

Lp(a) particles are heterogeneous low-density lipoprotein particlesexpressed predominantly in the liver (Witztum and Ginsberg, J Lipid Res.2016 March; 57(3):336-9). They are composed of Apolipoprotein(a) (Apo(a)or Lp(a) encoded by the LPA gene) linked to an LDL-like particle via theApoB poly-peptide. Genetically defined high Lp(a) particle serum levelsare unaffected by diet and exercise and are associated to increased riskto suffer from cardiovascular disease through the associatedatherosclerotic potential (Alonso et al., Journal of the AmericanCollege of Cardiology Vol. 63, No. 19, 2014). In terms of diagnosticsand preventive medicine the patient's serum level of Lp(a) particles isa highly prevalent, independent, genetic risk factor for coronary heartdisease and aortic stenosis (Saeedi and Frohlich Clinical Diabetes andEndocrinology (2016) 2:7). There is no current approved specific Lp(a)particle reduction therapy beyond indirect standard general LDL-loweringmeasures. Accordingly, methods for effective treatment, prevention andreduction of risk of suffering from disorders such as and associatedwith stroke, atherosclerosis, thrombosis and cardiovascular diseasessuch as coronary heart disease, aortic stenosis and other yetunidentified associated disorders, pathologies or syndromes arecurrently needed. The present invention addresses this unmet medicalneed.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a nucleic acid for inhibitingexpression of LPA in a cell, comprising at least one duplex region thatcomprises at least a portion of a first strand and at least a portion ofa second strand that is at least partially complementary to the firststrand, wherein said first strand is at least partially complementary toat least a portion of a RNA transcribed from the LPA gene, wherein saidfirst strand comprises a nucleotide sequence selected from the followingsequences: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41 or 43, wherein the nucleotides atpositions 2 and 14 from the 5′ end of the first strand are modified witha 2′ fluoro modification, and the nucleotides on the second strand whichcorrespond to positions 11-13 of the first strand are modified with a 2′fluoro modification.

The invention also provides a composition comprising the nucleic acid orconjugated nucleic acid of any aspect of the invention, and optionally aphysiologically acceptable excipient.

One aspect relates to a nucleic acid that is capable of inhibitingexpression of LPA for use as a medicament.

Also provided is a nucleic acid or conjugated nucleic acid according toany aspect of the invention for use in the treatment of a disease,disorder or syndrome and/or in the manufacture of a medicament fortreating a disease, disorder, or syndrome.

The invention provides a method of treating or preventing a disease,disorder or syndrome comprising administration of a compositioncomprising a nucleic acid or conjugated nucleic acid according to anyaspect of the invention to an individual in need of treatment. Thenucleic acid or conjugated nucleic acid may be administered to thesubject subcutaneously, intravenously or using any other applicationroutes such as oral, rectal or intraperitoneal.

A method of making the nucleic acid or conjugated nucleic acid accordingto the invention is also included.

The nucleic acid or the composition comprising the nucleic acid orconjugated nucleic acid of the invention may be used in the treatment ofa disease, disorder or syndrome. The treatment may be to prevent andreduce risk to suffer from stroke, atherosclerosis, thrombosis orcardiovascular diseases such as coronary heart disease or aorticstenosis and any other disease or pathology associated to elevatedlevels of Lp(a)-containing particles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid which is double strandedand directed to an expressed RNA transcript of LPA and compositionsthereof. These nucleic acids or conjugated nucleic acids can be used inthe treatment and prevention of a variety of diseases, disorders andsyndromes where reduced expression of LPA gene product is desirable.

One aspect relates to a nucleic acid for inhibiting expression of LPA ina cell, comprising at least one duplex region that comprises at least aportion of a first strand and at least a portion of a second strand thatis at least partially complementary to the first strand, wherein saidfirst strand is at least partially complementary to at least a portionof a RNA transcribed from the LPA gene, wherein said first strandcomprises, or preferably consists of, a nucleotide sequence selectedfrom the following sequences: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43, wherein thenucleotides at positions 2 and 14 from the 5′ end of the first strandare modified with a 2′ fluoro modification, and the nucleotides on thesecond strand which correspond to positions 11-13 of the first strandare modified with a 2′ fluoro modification.

One aspect relates to a nucleic acid for inhibiting expression of LPA ina cell, comprising at least one duplex region that comprises a firststrand and a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of a RNA transcribed from the LPAgene, wherein said first strand comprises, or preferably consists of, anucleotide sequence selected from the following sequences: SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, or 43, wherein the nucleotides at positions 2 and 14 from the 5′end of the first strand are modified with a 2′ fluoro modification, andthe nucleotides on the second strand which correspond to positions 11-13of the first strand are modified with a 2′ fluoro modification.

Such nucleic acids are able to efficiently reduce the expression of LPAin a cell and are very stable. The nucleic acid of the invention ispreferably capable of inhibiting expression of LPA in a cell to asimilar, such as the same, or a higher degree than the same nucleicacids with a different modification pattern in comparable conditions.More specifically, the nucleic acid of the invention is preferablycapable of inhibiting expression of LPA in a cell by 80, 90, 100, 105,110 or more percent as compared to the same nucleic acid with adifferent modification pattern in comparable conditions.

One aspect relates to a nucleic acid wherein all nucleotides of thenucleic acid are modified at the 2′ position of the sugar.

One aspect relates to a nucleic acid wherein the nucleic acid ismodified preferably along the entire length of the first strand withalternating 2′ O-methyl modifications and 2′ fluoro modifications.

One aspect relates to a nucleic acid wherein the remaining modificationsof the second strand are naturally occurring modifications, preferably2′ O-methyl. In other words, nucleotides on the second strand whichcorrespond to positions 11-13 of the first strand are modified with a 2′fluoro modification and all other nucleotides of the second strand aremodified with a naturally occurring modification, which is preferably 2′O-methyl.

The second strand may comprise a nucleotide sequence of SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, or44.

The nucleic acid may: a) be blunt ended at both ends; b) have anoverhang at one end and a blunt end at the other; or c) have an overhangat both ends. The nucleic acid is preferably blunt ended at both ends.

These nucleic acids among others have the advantage of being active invarious species that are relevant for pre-clinical and clinicaldevelopment and/or of having few relevant off-target effects as well asbeing stable in vivo and having a long duration of action. They alsocomprise comprises relatively few non-naturally occurring modifiednucleotides but are nonetheless able to efficiently inhibit the targetgene for long periods of time. The specific modification pattern withfew non-naturally occurring modified nucleotides (2′F modifiednucleotides) also makes them easier to synthesise.

The nucleic acid may comprise a first strand that comprises orpreferably consists of a nucleotide sequence of SEQ ID NO: 9, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 10; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 5, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 6; or a first strand that comprises orpreferably consists of a nucleotide sequence of SEQ ID NO: 1, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 2; or a first strand that comprises orpreferably consists of a nucleotide sequence of SEQ ID NO:3, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 4; or a first strand that comprises orpreferably consists of a nucleotide sequence of SEQ ID NO: 7, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 8; or a first strand that comprises orpreferably consists of a nucleotide sequence of SEQ ID NO: 11, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 12; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 13, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 14; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 15, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 16; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 17, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 18; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 19, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 20; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 21, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 22; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO:23, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 24; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO:25, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 26; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 27, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 28; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 29, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 30; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 31, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 32; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 33, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 34; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 35, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 36; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 37, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 38; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 39, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 40; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 41, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 42; or a first strand that comprisesor preferably consists of a nucleotide sequence of SEQ ID NO: 43, andoptionally a second strand that comprises or preferably consists of anucleotide sequence of SEQ ID NO: 44.

The nucleic acid wherein said first strand comprises, and preferablyconsists of, the nucleotide sequence of SEQ ID NO: 9 and optionally,wherein said second strand comprises, and preferably consists of, thenucleotide sequence of SEQ ID NO. 10.

The LPA gene comprises highly repetitive sequences. First strand nucleicacids with very similar sequences can therefore have perfect sequencecomplementarity to very different target regions of the mRNA.

One aspect is a nucleic acid for inhibiting expression of LPA in a cell,wherein the nucleic acid comprises at least one duplex region thatcomprises: a first strand; and a second strand, wherein said secondstrand is at least partially complementary to the first strand, whereinsaid first strand comprises a sequence of at least 15, preferably atleast 16, more preferably at least 17, yet more preferably at least 18and most preferably at least 19 nucleotides of any one of the referencesequences SEQ ID NO: 9, 5, 1, 3, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, or 43, and wherein the number of singlenucleotide mismatches and/or deletions and/or insertions in the firststrand sequence relative to the portion of the reference sequence thatis comprised in the first strand sequence is at most three, preferablyat most two, more preferably at most one and most preferably zero. Thesequence may however be modified by a number of modifications that donot change the identity of the nucleotide. For examples, modificationsof the backbone of the nucleic acid do not change the identity of thenucleotide because the base itself remains the same as in the referencesequence.

In one aspect, the first strand of the nucleic acid comprises a sequenceof at least 18 nucleotides of any one of the reference sequences,preferably of any one of the reference sequences SEQ ID NO: 9 and 5, andwherein the number of single-nucleotide mismatches and/or deletionsand/or insertions in the first strand sequence relative to the portionof the reference sequence that is comprised in the first strand sequenceis at most one, and preferably zero.

In one aspect, the first strand of the nucleic acid comprises a sequenceof at least 19 nucleotides of any of the reference sequences SEQ ID NO:9 and 5.

When reference is made herein to a reference sequence comprising orconsisting of unmodified nucleotides, this reference is not limited tothe sequence with unmodified nucleotides. The same reference alsoencompasses the same nucleotide sequence in which one, several, such astwo, three, four, five, six, seven or more, including all, nucleotidesare modified by modifications such as 2′-OMe, 2′-F, a ligand, a linker,a 3′ end or 5′ end modification or any other modification. It alsorefers to sequences in which two or more nucleotides are linked to eachother by the natural phosphodiester linkage or by any other linkage suchas a phosphorothioate or a phosphorodithioate linkage.

A double-stranded nucleic acid is a nucleic acid in which the firststrand and the second strand hybridise to each other over at least partof their lengths and are therefore capable of forming a duplex regionunder physiological conditions, such as in PBS at 37° C. at aconcentration of 1 μM of each strand. The first and second strand arepreferably able to hybridise to each other and therefore to form aduplex region over a region of at least 15 nucleotides, preferably 16,17, 18 or 19 nucleotides. This duplex region comprises nucleotide baseparings between the two strands, preferably based on Watson-Crick basepairing and/or wobble base pairing (such as GU base pairing). All thenucleotides of the two strands within a duplex region do not have tobase pair to each other to form a duplex region. A certain number ofmismatches, deletions or insertions between the nucleotide sequences ofthe two strands are acceptable. Overhangs on either end of the first orsecond strand or unpaired nucleotides at either end of thedouble-stranded nucleic acid are also possible. The double strandednucleic acid is preferably a stable double stranded nucleic acid underphysiological conditions and preferably has a melting temperature (Tm)of 45° C. or more, preferably 50° C. or more, and more preferably 55° C.or more for example in PBS at a concentration 1 μM of each first strandand the second strand are preferably capable of forming a duplex region(ie are complementary to each other) over i) at least a portion of theirlengths, preferably over at least 15 nucleotides of both of theirlengths, ii) over the entire length of the first strand, iii) over theentire length of the second strand and/or iv) over the entire length ofboth the first and the second strand. Strands being complementary toeach other over a certain length means that the strands are able to basepair to each other, either via Watson-Crick or wobble base pairing, overthat length. Each nucleotide of the length does not necessarily have tobe able to base pair with its counterpart in the other strand over theentire given length as long as a stable double-stranded nucleotide underphysiological conditions can be formed. This is however preferred.

A certain number of mismatches, deletions or insertions between thefirst (antisense) strand and the target sequence, or between the firststrand and the second (sense) strand can be tolerated in the context ofsiRNA and even have the potential in certain cases to increase activity.

By nucleic acid it is meant a nucleic acid comprising two strandscomprising nucleotides, that is able to interfere with gene expression.Inhibition may be complete or partial and results in down regulation ofgene expression in a targeted manner. The nucleic acid comprises twoseparate polynucleotide strands; the first strand, which may also be aguide strand; and a second strand, which may also be a passenger strand.The first strand and the second strand may be part of the samepolynucleotide molecule that is self-complementary which ‘folds’ back toform a double stranded molecule. The nucleic acid may be an siRNAmolecule.

The nucleic acid may comprise ribonucleotides, modified ribonucleotides,deoxynucleotides, deoxyribonucleotides, or nucleotide analoguesnon-nucleotides that are able to mimic nucleotides such that they may‘pair’ with the corresponding base on the target sequence orcomplementary strand. The nucleic acid may further comprise adouble-stranded nucleic acid portion or duplex region formed by all or aportion of the first strand (also known in the art as a guide strand)and all or a portion of the second strand (also known in the art as apassenger strand). The duplex region is defined as beginning with thefirst base pair formed between the first strand and the second strandand ending with the last base pair formed between the first strand andthe second strand, inclusive.

By duplex region it is meant the region in two complementary orsubstantially complementary oligonucleotides that form base pairs withone another, either by Watson-Crick base pairing or any other mannerthat allows for a duplex between oligonucleotide strands that arecomplementary or substantially complementary. For example, anoligonucleotide strand having 21 nucleotide units can base pair withanother oligonucleotide of 21 nucleotide units, yet only 19 nucleotideson each strand are complementary or substantially complementary, suchthat the “duplex region” consists of 19 base pairs. The remaining basepairs may exist as 5′ and 3′ overhangs, or as single stranded regions.Further, within the duplex region, 100% complementarity is not required;substantial complementarity is allowable within a duplex region.Substantial complementarity refers to complementarity between thestrands such that they are capable of annealing under biologicalconditions. Techniques to empirically determine if two strands arecapable of annealing under biological conditions are well known in theart. Alternatively, two strands can be synthesised and added togetherunder biological conditions to determine if they anneal to one another.

The portion of the first strand and second strand that form at least oneduplex region may be fully complementary or be at least partiallycomplementary to each other.

Depending on the length of a nucleic acid, a perfect match in terms ofbase complementarity between the first strand and the second strand isnot necessarily required. However, the first and second strands must beable to hybridise under physiological conditions.

The complementarity between the first strand and second strand in the atleast one duplex region may be perfect in that there are no nucleotidemismatches or additional/deleted nucleotides in either strand.Alternatively, the complementarity may not be perfect. Thecomplementarity may be from about 70% to about 100%. More specifically,the complementarity may be at least 70%, 75%, 80%, 85%, 90% or 95% andintermediate values.

In the context of this invention, “a portion of” as for example in “oneduplex region that comprises at least a portion of a first strand”should be understood to mean that the duplex region comprises at least10, preferably at least 12, more preferably at least 14, yet morepreferably at least 16, even more preferably at least 18 and mostpreferably all of the nucleotides of a given reference strand sequence.The portion of the reference sequence in the duplex region is at least70%, preferably at least 80%, more preferably at least 90%, yet morepreferably at least 95% and most preferably 100% identical to thecorresponding portion of the reference sequence. Alternatively, thenumber of single nucleotide mismatches relative to the portion of thereference sequence is at most three, preferably at most two, morepreferably at most one and most preferably zero.

The first strand and the second strand may each comprise a region ofcomplementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the sequenceslisted in Table 1.

Use of a nucleic acid according to the present invention involves theformation of a duplex region between all or a portion of the firststrand and a portion of a target nucleic acid. The portion of the targetnucleic acid that forms a duplex region with the first strand, definedas beginning with the first base pair formed between the first strandand the target sequence and ending with the last base pair formedbetween the first strand and the target sequence, inclusive, is thetarget nucleic acid sequence or simply, target sequence. The duplexregion formed between the first strand and the second strand need not bethe same as the duplex region formed between the first strand and thetarget sequence. That is, the second strand may have a sequencedifferent from the target sequence; however, the first strand must beable to form a duplex structure with both the second strand and thetarget sequence, at least under physiological conditions.

The complementarity between the first strand and the target sequence maybe perfect (no nucleotide mismatches or additional/deleted nucleotidesin either nucleic acid).

The complementarity between the first strand and the target sequence maynot be perfect. The complementarity may be from about 70% to about 100%.More specifically, the complementarity may be at least 70%, 80%, 85%,90% or 95% and intermediate values.

The identity between the first strand and the complementary sequence ofthe target sequence may range from about 75% to about 100%. Morespecifically, the complementarity may be at least 75%, 80%, 85%, 90% or95% and intermediate values, provided a nucleic acid is capable ofreducing or inhibiting the expression of LPA.

A nucleic acid having less than 100% complementarity between the firststrand and the target sequence may be able to reduce the expression ofLPA to the same level as a nucleic acid having perfect complementaritybetween the first strand and target sequence. Alternatively, it may beable to reduce expression of LPA to a level that is 15%-100% of thelevel of reduction achieved by the nucleic acid with perfectcomplementarity.

In one aspect, the nucleic acid comprises a first nucleic acid strandand a second nucleic acid strand, wherein the first strand is capable ofhybridising under physiological conditions to a nucleic acid of sequenceSEQ ID NO: 10, 6, 2, 4, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, or 44;

wherein the second strand is capable of hybridising under physiologicalconditions to the first strand to form a duplex region; and

wherein the nucleotides at positions 2 and 14 from the 5′ end of thefirst strand are modified with a 2′ fluoro modification, and thenucleotides on the second strand which correspond to positions 11-13 ofthe first strand are modified with a 2′ fluoro modification.

Nucleic acids that are capable of hybridising under physiologicalconditions are nucleic acids that are capable of forming base pairs,preferably Watson-Crick or wobble base-pairs, between at least a portionof the opposed nucleotides in the strands so as to form at least aduplex region. Such a double-stranded nucleic acid is preferably astable double-stranded nucleic acid under physiological conditions (forexample in PBS at 37° C. at a concentration of 1 μM of each strand),meaning that under such conditions, the two strands stay hybridised toeach other. The Tm of the double-stranded nucleotide is preferably 45°C. or more, preferably 50° C. or more and more preferably 55° C. ormore.

One aspect relates to a nucleic acid for inhibiting expression of LPA,wherein the nucleic acid comprises a first sequence of at least 15,preferably at least 16, more preferably at least 17, yet more preferablyat least 18 and most preferably all nucleotides differing by no morethan 3 nucleotides, preferably no more than 2 nucleotides, morepreferably no more than 1 nucleotide and most preferably not differingby any nucleotide from any of the sequences of Table 1, the firstsequence being able to hybridise to a target gene transcript (such as anmRNA) under physiological conditions. Preferably the nucleic acidfurther comprises a second sequence of at least 15, preferably at least16, more preferably at least 17, yet more preferably at least 18 andmost preferably all nucleotides differing by no more than 3 nucleotides,preferably no more than 2 nucleotides, more preferably no more than 1nucleotide and most preferably not differing by any nucleotide from anyof the sequences of Table 1, the second sequence being able to hybridiseto the first sequence under physiological conditions and preferably thenucleic acid being an siRNA that is capable of inhibiting LPA expressionvia the RNAi pathway.

The nucleic acids described herein may be capable of inhibiting theexpression of LPA. Inhibition may be complete, i.e. 0% remainingexpression compared of the expression level of LPA in the absence of thenucleic acid of the invention. Inhibition of LPA expression may bepartial, i.e. it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95% or intermediate values of LPA expression in the absence ofa nucleic acid of the invention. The level of inhibition may be measuredby comparing a treated sample with an untreated sample or with a sampletreated with a control such as for example a siRNA that does not targetLPA. Inhibition may be measured by measuring LPA mRNA and/or proteinlevels or levels of a biomarker or indicator that correlates with LPApresence or activity. It may be measured in cells that may have beentreated in vitro with a nucleic acid described herein. Alternatively, orin addition, inhibition may be measured in cells, such as hepatocytes,or tissue, such as liver tissue, or an organ, such as the liver, or in abody fluid such as blood, serum, lymph or any other body part that hasbeen taken from a subject previously treated with a nucleic aciddisclosed herein. Preferably inhibition of LPA expression is determinedby comparing the LPA mRNA level measured in LPA-expressing cells after24 or 48 hours in vitro treatment under ideal conditions (see theexamples for appropriate concentrations and conditions) with a nucleicacid disclosed herein to the LPA mRNA level measured in the same cellsthat were untreated or mock treated or treated with a control nucleicacid.

As used herein, the term “inhibit”, “down-regulate”, or “reduce” withrespect to gene expression means the expression of the gene, or level ofRNA molecules or equivalent RNA molecules encoding one or more proteinsor protein subunits (e.g., mRNA), or activity of one or more proteins orprotein subunits, is reduced below that observed in the absence of thenucleic acid or conjugated nucleic acid of the invention or in referenceto an siRNA molecule with no known homology to human transcripts (hereintermed non-silencing control). Such control may be conjugated andmodified in an analogous manner to the molecule of the invention anddelivered into the target cell by the same route; for example theexpression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,15%, or to intermediate values, or less than that observed in theabsence of the nucleic acid or conjugated nucleic acid or in thepresence of a non-silencing control.

The nucleic acid may comprise a first strand and a second strand thatare each from 19-25 nucleotides in length. The first strand and thesecond strand may be of different lengths.

The first strand and/or the second strand may each be from 17-35,preferably 18-30, more preferably 19-25 and most preferably 19nucleotides in length and at least one duplex region may be from 10-25nucleotides, preferably 18-23 nucleotides in length. The duplex maycomprise two separate strands or it may comprise a single strand whichcomprises the first strand and the second strand.

The first strand may be 17-25 nucleotides in length, preferably it maybe 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24nucleotides in length. Most preferably, the first strand is 19nucleotides in length. The second strand may independently be 17-25nucleotides in length, preferably it may be 18-24 nucleotides in length,it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. Morepreferably, the second strand is 18 or 19 nucleotides in length, andmost preferably it is 18 nucleotides in length.

The nucleic acid may be 15-25 nucleotide pairs in length. The nucleicacid may be 17-23 nucleotide pairs in length. The nucleic acid may be17-25 nucleotide pairs in length. The nucleic acid may be 23-24nucleotide pairs in length. The nucleic acid may be 19-21 nucleotidepairs in length. The nucleic acid may be 21-23 nucleotide pairs inlength.

The nucleic acid may comprise a duplex region that consists of 19-25nucleotide base pairs. The duplex region may consist of 17, 18, 19, 20,21, 22, 23, 24 or 25 base pairs, which may be contiguous. Preferably,the duplex region consists of 19 base pairs.

Preferably, the nucleic acid mediates RNA interference.

In one embodiment, the nucleic acid for inhibiting expression of LPA ina cell, comprises at least one duplex region that comprises a firststrand and a second strand that is at least partially complementary tothe first strand, wherein said first strand comprises a sequence of atleast 15, preferably at least 16, more preferably at least 17, yet morepreferably at least 18 and most preferably at least 19 nucleotides witha sequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, yet more preferably at least 95% and mostpreferably 100% of any of sequences SEQ ID NOs: 9, 5, 1, 3, 7, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43.

In a further aspect the nucleic acid or conjugated nucleic acid asdescribed may reduce the expression of LPA by at least 15% compared tothe expression observed in the absence of the nucleic acid or conjugatednucleic acid. All preferred features of any of the previous aspects alsoapply to this aspect. In particular, the expression of LPA may bereduced to at least the following given % or less than 90%, 80%, 70%,60%, 50%, 40%, 30%, 20%, 15% or less, and intermediate values, than thatobserved in the absence of the nucleic acid or conjugated nucleic acidor in the presence of a non-silencing control.

The nucleic acid may have an overhang at one end and a blunt end at theother. The nucleic acid may have an overhang at both ends. The nucleicacid may be blunt ended at both ends. The nucleic acid may be bluntended at the end with the 5′-end of the first strand and the 3′-end ofthe second strand or at the 3′-end of the first strand and the 5′-end ofthe second strand.

An “overhang” as used herein has its normal and customary meaning in theart, i.e. a single stranded portion of a nucleic acid that extendsbeyond the terminal nucleotide of a complementary strand in a doublestrand nucleic acid. The term “blunt end” includes double strandednucleic acid whereby both strands terminate at the same position,regardless of whether the terminal nucleotide(s) are base-paired. Theterminal nucleotide of a first strand and a second strand at a blunt endmay be base paired. The terminal nucleotide of a first strand and asecond strand at a blunt end may not be paired. The terminal twonucleotides of a first strand and a second strand at a blunt end may bebase-paired. The terminal two nucleotides of a first strand and a secondstrand at a blunt end may not be paired.

The nucleic acid may comprise an overhang at a 3′- or 5′-end. Thenucleic acid may have a 3′-overhang on the first strand. The nucleicacid may have a 3′-overhang on the second strand. The nucleic acid mayhave a 5′-overhang on the first strand. The nucleic acid may have a5′-overhang on the second strand. The nucleic acid may have an overhangat both the 5′-end and 3′-end of the first strand. The nucleic acid mayhave an overhang at both the 5′-end and 3′-end of the second strand. Thenucleic acid may have a 5′ overhang on the first strand and a 3′overhang on the second strand. The nucleic acid may have a 3′ overhangon the first strand and a 5′ overhang on the second strand. The nucleicacid may have a 3′ overhang on the first strand and a 3′ overhang on thesecond strand. The nucleic acid may have a 5′ overhang on the firststrand and a 5′ overhang on the second strand.

An overhang at the 3′-end or 5′ end of the second strand or the firststrand may be selected from consisting of 1, 2, 3, 4 and 5 nucleotidesin length. Optionally, an overhang may consist of 1 or 2 nucleotides,which may or may not be modified.

Preferably, the nucleic acid is an siRNA. siRNAs are short interferingor short silencing RNAs that are able to inhibit the expression of atarget gene through the RNA interference (RNAi) pathway. Inhibitionoccurs through targeted degradation of mRNA transcripts of the targetgene after transcription. The siRNA forms part of the RISC complex. TheRISC complex specifically targets the target RNA by sequencecomplementarity of the first (antisense) strand with the targetsequence.

Preferably, the nucleic acid mediates RNA interference (RNAi). Thenucleic acid, or at least the first strand of the nucleic acid, istherefore preferably able to be incorporated into the RISC complex. As aresult, the nucleic acid, or at least the first strand of the nucleicacid, is therefore able to guide the RISC complex to a specific targetRNA with which the nucleic acid, or at least the first strand of thenucleic acid, is at least partially complementary. The RISC complex thenspecifically cleaves this target RNA and as a result leads to inhibitionof the expression of the gene from which the RNA stems.

Nucleic Acid Modifications

Unmodified polynucleotides, particularly ribonucleotides, may be proneto degradation by cellular nucleases, and, as such,modifications/modified nucleotides may be included in the nucleic acidof the invention. Such modifications may help to stabilise the nucleicacid by making them more resistant against nucleases. This improvedresistance allows nucleic acids to be active in mediating RNAinterference for longer time periods and is especially desirable whenthe nucleic acids are to be used for treatment.

Modifications of the nucleic acid of the present invention generallyprovide a powerful tool in overcoming potential limitations including,but not limited to, in vitro and in vivo stability and bioavailabilityinherent to native RNA molecules. The nucleic acid according to theinvention may be modified by chemical modifications. Modified nucleicacid can also minimise the possibility of inducing interferon activityin humans. Modification can further enhance the functional delivery of anucleic acid to a target cell. The modified nucleic acid of the presentinvention may comprise one or more chemically modified ribonucleotidesof either or both of the first strand or the second strand. Aribonucleotide may comprise a chemical modification of the base, sugaror phosphate moieties. The ribonucleic acid may be modified bysubstitution or insertion with analogues of nucleic acids or bases.

Preferably, at least one nucleotide of the first and/or second strand ofthe nucleic acid is a modified nucleotide, preferably a non-naturallyoccurring nucleotide such as preferably a 2′-F modified nucleotide.

One or more nucleotides of a nucleic acid of the present invention maybe modified. The nucleic acid may comprise at least one modifiednucleotide. The modified nucleotide may be in the first strand. Themodified nucleotide may be in the second strand. The modified nucleotidemay be in the duplex region. The modified nucleotide may be outside theduplex region, i.e., in a single stranded region. The modifiednucleotide may be on the first strand and may be outside the duplexregion. The modified nucleotide may be on the second strand and may beoutside the duplex region. The 3′-terminal nucleotide of the firststrand may be a modified nucleotide. The 3′-terminal nucleotide of thesecond strand may be a modified nucleotide. The 5′-terminal nucleotideof the first strand may be a modified nucleotide. The 5′-terminalnucleotide of the second strand may be a modified nucleotide.

A nucleic acid of the invention may have 1 modified nucleotide or anucleic acid of the invention may have about 2-4 modified nucleotides,or a nucleic acid may have about 4-6 modified nucleotides, about 6-8modified nucleotides, about 8-10 modified nucleotides, about 10-12modified nucleotides, about 12-14 modified nucleotides, about 14-16modified nucleotides about 16-18 modified nucleotides, about 18-20modified nucleotides, about 20-22 modified nucleotides, about 22-24modified nucleotides, 24-26 modified nucleotides or about 26-28 modifiednucleotides or all of the nucleotides may be modified. In each case thenucleic acid comprising said modified nucleotides retains at least 50%of its activity as compared to the same nucleic acid but without saidmodified nucleotides or vice versa. The nucleic acid may retain 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and intermediate valuesof its activity as compared to the same nucleic acid but without saidmodified nucleotides, or may have more than 100% of the activity of thesame nucleic acid without said modified nucleotides.

The modified nucleotide may be a purine or a pyrimidine. At least halfof the purines may be modified. At least half of the pyrimidines may bemodified. All of the purines may be modified. All of the pyrimidines maybe modified. The modified nucleotides may be selected from the groupconsisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methylmodified nucleotide, a 2′ modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, a non-natural base comprisingnucleotide, a nucleotide comprising a 5′-phosphorothioate group, anucleotide comprising a 5′ phosphate or 5′ phosphate mimic and aterminal nucleotide linked to a cholesteryl derivative or a dodecanoicacid bisdecylamide group.

The nucleic acid may comprise a nucleotide comprising a modifiednucleotide, wherein the base is selected from 2-aminoadenosine,2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil,dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g.,5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

Nucleic acids discussed herein include unmodified RNA as well as RNAwhich has been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, for example as occur naturally in the human body. Modifiednucleotide as used herein refers to a nucleotide in which one or more ofthe components of the nucleotides, namely sugars, bases, and phosphatemoieties, are different from those which occur in nature. While they arereferred to as modified nucleotides they will of course, because of themodification, the term also includes molecules which are notnucleotides, for example a polynucleotide molecule in which theribophosphate backbone is replaced with a non-ribophosphate constructthat allows hybridisation between strands i.e. the modified nucleotidesmimic the ribophosphate backbone.

Many of the modifications described below that occur within a nucleicacid will be repeated within a polynucleotide molecule, such as amodification of a base, or a phosphate moiety, or a non-linking 0 of aphosphate moiety. In some cases the modification will occur at all ofthe possible positions/nucleotides in the polynucleotide but in manycases it will not. A modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal regions, such as at a position ona terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a nucleic acid of the invention or may only occur in asingle strand region of a nucleic acid of the invention. Aphosphorothioate modification at a non-linking 0 position may only occurat one or both termini, may only occur in a terminal region, e.g., at aposition on a terminal nucleotide or in the last 2, 3, 4 or 5nucleotides of a strand, or may occur in duplex and/or in single strandregions, particularly at termini. The 5′ end or 3′ ends may bephosphorylated.

Stability of a nucleic acid of the invention may be increased byincluding particular bases in overhangs, or to include modifiednucleotides, in single strand overhangs, e.g., in a 5′ or 3′ overhang,or in both. Purine nucleotides may be included in overhangs. All or someof the bases in a 3′ or 5′ overhang may be modified. Modifications caninclude the use of modifications at the 2′ OH group of the ribose sugar,the use of deoxyribonucleotides, instead of ribonucleotides, andmodifications in the phosphate group, such as phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

Nucleases can hydrolyse nucleic acid phosphodiester bonds. However,chemical modifications to nucleic acids can confer improved properties,and, can render oligoribonucleotides more stable to nucleases.

Modified nucleic acids, as used herein, can include one or more of:

-   (i) alteration, e.g., replacement, of one or both of the non-linking    phosphate oxygens and/or of one or more of the linking phosphate    oxygens (referred to as linking even if at the 5′ and 3′ terminus of    the nucleic acid of the invention);-   (ii) alteration, e.g., replacement, of a constituent of the ribose    sugar, e.g., of the 2′ hydroxyl on the ribose sugar;-   (iii) replacement of the phosphate moiety with “dephospho” linkers;-   (iv) modification or replacement of a naturally occurring base;-   (v) replacement or modification of the ribose-phosphate backbone;-   (vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,    modification or replacement of a terminal phosphate group or    conjugation of a moiety, e.g., a fluorescently labelled moiety, to    either the 3′ or 5′ end of RNA.

The terms replacement, modification, alteration, indicate a differencefrom a naturally occurring molecule.

Specific modifications are discussed in more detail below.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulphur. One, each or both non-linking oxygens in thephosphate group can be independently any one of S, Se, B, C, H, N, or OR(R is alkyl or aryl).

The phosphate linker can also be modified by replacement of a linkingoxygen with nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at a terminal oxygen. Replacement of thenon-linking oxygens with nitrogen is possible.

A modified nucleotide can include modification of the sugar groups. The2′ hydroxyl group (OH) can be modified or replaced with a number ofdifferent “oxy” or “deoxy” substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleicacids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylenebridge, to the 4′ carbon of the same ribose sugar; 0-AMINE (AMINE=NH2;alkylamino, dialkylamino, heterocyclyl, arylamino, diary) amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino)and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino,dialkylamino, heterocyclyl, arylamino, diary) amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino).

“Deoxy” modifications include hydrogen, halogen, amino (e.g., NH2;alkylamino, dialkylamino, heterocyclyl, arylamino, diary) amino,heteroaryl amino, diheteroaryl amino, or amino acid);NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl,cycloalkyl, aryl, alkenyl and alkynyl, which may be optionallysubstituted with e.g., an amino functionality. Other substitutents ofcertain embodiments include 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl,2′-C-allyl, and 2′-fluoro. The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a modified nucleotide maycontain a sugar such as arabinose.

Modified nucleotides can also include “abasic” sugars, which lack anucleobase at C-1′. These abasic sugars can further containmodifications at one or more of the constituent sugar atoms.

The 2′ modifications may be used in combination with one or morephosphate linker modifications (e.g., phosphorothioate).

The phosphate groups can individually be replaced by non-phosphoruscontaining connectors.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.In certain embodiments, replacements may include themethylenecarbonylamino and methylenemethylimino groups.

The phosphate linker and ribose sugar may be replaced by nucleaseresistant nucleotides.

Examples include the morpholino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNAsurrogates may be used.

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end or the 5′ end or both ends of themolecule. They can include modification or replacement of an entireterminal phosphate or of one or more of the atoms of the phosphategroup. For example, the 3′ and 5′ ends of an oligonucleotide can beconjugated to other functional molecular entities such as labellingmoieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 orCy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron orester). The functional molecular entities can be attached to the sugarthrough a phosphate group and/or a linker. The terminal atom of thelinker can connect to or replace the linking atom of the phosphate groupor the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, thelinker can connect to or replace the terminal atom of a nucleotidesurrogate (e.g., PNAs). These spacers or linkers can include e.g.,—(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—,—(CH₂CH₂O)_(n)CH₂CH₂O— (e.g., n=3 or 6), abasic sugars, amide, carboxy,amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide,or morpholino, or biotin and fluorescein reagents. The 3′ end can be an—OH group.

Other examples of terminal modifications include dyes, intercalatingagents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases, EDTA, lipophilic carriers (e.g., cholesterol, cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O (hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g., biotin), transport/absorption facilitators (e.g.,aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,imidazole, bisimidazole, histamine, imidazole clusters,acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingto modulate activity or to modulate resistance to degradation. Terminalmodifications useful for modulating activity include modification of the5′ end with phosphate or phosphate analogues. Nucleic acids of theinvention, on the first or second strand, may be 5′ phosphorylated orinclude a phosphoryl analogue at the 5′ prime terminus. 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)₂(S)P—O-5′); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.,5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g., RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′vinylphosphonate,5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂—),ethoxymethyl, etc., e.g., RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the groups to be added may include fluorophores, e.g.,fluorescein or an Alexa dye. Terminal modifications can also be usefulfor enhancing uptake, useful modifications for this include cholesterol.Terminal modifications can also be useful for cross-linking an RNA agentto another moiety.

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNAs havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogues of any of the abovebases and “universal bases” can be employed. Examples include2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N<4>-acetyl cytosine,2-thiocytosine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases.

As used herein, the terms “non-pairing nucleotide analogue” means anucleotide analogue which includes a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analogue is aribonucleotide. In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes withoutlimitation a halogen, alcohol, amine, carboxylic, ester, amide,aldehyde, ketone, ether groups.

Certain moieties may be linked to the 5′ terminus of the first strand orthe second strand. These include abasic ribose moiety, abasicdeoxyribose moiety, modifications abasic ribose and abasic deoxyribosemoieties including 2′ O alkyl modifications; inverted abasic ribose andabasic deoxyribose moieties and modifications thereof, C6-imino-Pi; amirror nucleotide including L-DNA and L-RNA; 5′OMe nucleotide; andnucleotide analogues including 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non-bridging methylphosphonate and 5′-mercaptomoieties.

The nucleic acid of the present invention may comprise an abasicnucleotide. The term “abasic” as used herein, refers to moieties lackinga base or having other chemical groups in place of a base at the 1′position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribosederivative.

The nucleic acid may comprise one or more nucleotides on the secondand/or first strands that are modified. Alternating nucleotides may bemodified, to form modified nucleotides.

Alternating as described herein means to occur one after another in aregular way. In other words, alternating means to occur in turnrepeatedly. For example, if one nucleotide is modified, the nextcontiguous nucleotide is not modified and the following contiguousnucleotide is modified and so on. One nucleotide may be modified with afirst modification, the next contiguous nucleotide may be modified witha second modification and the following contiguous nucleotide ismodified with the first modification and so on, where the first andsecond modifications are different.

One or more of the odd numbered nucleotides of the first strand of thenucleic acid of the invention may be modified wherein the first strandis numbered 5′ to 3′, the 5′-most nucleotide being nucleotide number 1of the first strand. The term “odd numbered” as described herein means anumber not divisible by two. Examples of odd numbers are 1, 3, 5, 7, 9,11 and so on. One or more of the even numbered nucleotides of the firststrand of the nucleic acid of the invention may be modified, wherein thefirst strand is numbered 5′ to 3′. The term “even numbered” as describedherein means a number which is evenly divisible by two. Examples of evennumbers are 2, 4, 6, 8, 10, 12, 14 and so on. One or more of the oddnumbered nucleotides of the second strand of the nucleic acid of theinvention may be modified wherein the second strand is numbered 3′ to5′, the 3′-most nucleotide being nucleotide number 1 of the secondstrand. One or more of the even numbered nucleotides of the secondstrand of the nucleic acid of the invention may be modified, wherein thesecond strand is numbered 3′ to 5′.

One or more nucleotides on the first and/or second strand may bemodified, to form modified nucleotides. One or more of the odd numberednucleotides of the first strand may be modified. One or more of the evennumbered nucleotides of the first strand may be modified by at least asecond modification, wherein the at least second modification isdifferent from the modification on the one or more odd nucleotides. Atleast one of the one or more modified even numbered nucleotides may beadjacent to at least one of the one or more modified odd numberednucleotides.

A plurality of odd numbered nucleotides in the first strand may bemodified in the nucleic acid of the invention. A plurality of evennumbered nucleotides in the first strand may be modified by a secondmodification. The first strand may comprise adjacent nucleotides thatare modified by a common modification. The first strand may alsocomprise adjacent nucleotides that are modified by a second differentmodification.

One or more of the odd numbered nucleotides of the second strand may bemodified by a modification that is different to the modification of theodd numbered nucleotides on the first strand and/or one or more of theeven numbered nucleotides of the second strand may be modified by thesame modification of the odd numbered nucleotides of the first strand.At least one of the one or more modified even numbered nucleotides ofthe second strand may be adjacent to the one or more modified oddnumbered nucleotides. A plurality of odd numbered nucleotides of thesecond strand may be modified by a common modification and/or aplurality of even numbered nucleotides may be modified by the samemodification that is present on the first stand odd numberednucleotides. A plurality of odd numbered nucleotides on the secondstrand may be modified by a second modification, wherein the secondmodification is different from the modification of the first strand oddnumbered nucleotides.

The second strand may comprise adjacent nucleotides that are modified bya common modification, which may be a second modification that isdifferent from the modification of the odd numbered nucleotides of thefirst strand.

In the nucleic acid of the invention, each of the odd numberednucleotides in the first strand and each of the even numberednucleotides in the second strand may be modified with a commonmodification and, each of the even numbered nucleotides may be modifiedin the first strand with a second modification and each of the oddnumbered nucleotides may be modified in the second strand with a seconddifferent modification.

The nucleic acid of the invention may have the modified nucleotides ofthe first strand shifted by at least one nucleotide relative to theunmodified or differently modified nucleotides of the second strand.

One or more or each of the odd numbered nucleotides may be modified inthe first strand and one or more or each of the even numberednucleotides may be modified in the second strand. One or more or each ofthe alternating nucleotides on either or both strands may be modified bya second modification. One or more or each of the even numberednucleotides may be modified in the first strand and one or more or eachof the even numbered nucleotides may be modified in the second strand.One or more or each of the alternating nucleotides on either or bothstrands may be modified by a second modification. One or more or each ofthe odd numbered nucleotides may be modified in the first strand and oneor more of the odd numbered nucleotides may be modified in the secondstrand by a common modification. One or more or each of the alternatingnucleotides on either or both strands may be modified by a secondmodification. One or more or each of the even numbered nucleotides maybe modified in the first strand and one or more or each of the oddnumbered nucleotides may be modified in the second strand by a commonmodification. One or more or each of the alternating nucleotides oneither or both strands may be modified by a second modification.

The nucleic acid of the invention may comprise single or double strandedconstructs that comprise at least two regions of alternatingmodifications in one or both of the strands. These alternating regionscan comprise up to about 12 nucleotides but preferably comprise fromabout 3 to about 10 nucleotides. The regions of alternating nucleotidesmay be located at the termini of one or both strands of the nucleic acidof the invention. The nucleic acid may comprise from 4 to about 10nucleotides of alternating nucleotides at each termini (3′ and 5′) andthese regions may be separated by from about 5 to about 12 contiguousunmodified or differently or commonly modified nucleotides.

The odd numbered nucleotides of the first strand may be modified and theeven numbered nucleotides may be modified with a second modification.The second strand may comprise adjacent nucleotides that are modifiedwith a common modification, which may be the same as the modification ofthe odd numbered nucleotides of the first strand. One or morenucleotides of second strand may also be modified with the secondmodification. One or more nucleotides with the second modification maybe adjacent to each other and to nucleotides having a modification thatis the same as the modification of the odd numbered nucleotides of thefirst strand. The first strand may also comprise phosphorothioatelinkages between the two nucleotides at the 3′ end and at the 5′ end.The second strand may comprise a phosphorothioate linkage between thetwo nucleotides at 5′ end. The second strand may also be conjugated to aligand at the 5′ end.

The nucleic acid of the invention may comprise a first strand comprisingadjacent nucleotides that are modified with a common modification. Oneor more of such nucleotides may be adjacent to one or more nucleotideswhich may be modified with a second modification. One or morenucleotides with the second modification may be adjacent. The secondstrand may comprise adjacent nucleotides that are modified with a commonmodification, which may be the same as one of the modifications of oneor more nucleotides of the first strand. One or more nucleotides ofsecond strand may also be modified with the second modification. One ormore nucleotides with the second modification may be adjacent. The firststrand may also comprise phosphorothioate linkages between the twonucleotides at the 5′ end and at the 3′ end. The second strand maycomprise a phosphorothioate linkage between the two nucleotides at the3′ end. The second strand may also be conjugated to a ligand at the 5′end.

The nucleotides numbered from 5′ to 3′ on the first strand and 3′ to 5′on the second strand, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25may be modified by a modification on the first strand. The nucleotidesnumbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modifiedby a second modification on the first strand. The nucleotides numbered1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by amodification on the second strand. The nucleotides numbered 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a secondmodification on the second strand. Nucleotides are numbered for the sakeof the nucleic acid of the present invention from 5′ to 3′ on the firststrand and 3′ to 5′ on the second strand

The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24may be modified by a modification on the first strand. The nucleotidesnumbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by asecond modification on the first strand. The nucleotides numbered 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification onthe second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22 and 24 may be modified by a second modification on the secondstrand.

Clearly, if the first and/or the second strand are shorter than 25nucleotides in length, such as 19 nucleotides in length, there are nonucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified. Theskilled person understands the description above to apply to shorterstrands, accordingly.

One or more modified nucleotides on the first strand may be paired withmodified nucleotides on the second strand having a common modification.One or more modified nucleotides on the first strand may be paired withmodified nucleotides on the second strand having a differentmodification. One or more modified nucleotides on the first strand maybe paired with unmodified nucleotides on the second strand. One or moremodified nucleotides on the second strand may be paired with unmodifiednucleotides on the first strand. In other words, the alternatingnucleotides can be aligned on the two strands such as, for example, allthe modifications in the alternating regions of the second strand arepaired with identical modifications in the first strand or alternativelythe modifications can be offset by one nucleotide with the commonmodifications in the alternating regions of one strand pairing withdissimilar modifications (i.e. a second or further modification) in theother strand. Another option is to have dissimilar modifications in eachof the strands.

The modifications on the first strand may be shifted by one nucleotiderelative to the modified nucleotides on the second strand, such thatcommon modified nucleotides are not paired with each other.

The modification and/or modifications may each and individually beselected from the group consisting of 3′-terminal deoxy-thymine,2′-O-methyl, a 2′-deoxy-modification, a 2′-amino-modification, a2′-alkyl-modification, a morpholino modification, a phosphoramidatemodification, 5′-phosphorothioate group modification, a 5′ phosphate or5′ phosphate mimic modification and a cholesteryl derivative or adodecanoic acid bisdecylamide group modification and/or the modifiednucleotide may be any one of a locked nucleotide, an abasic nucleotideor a non-natural base comprising nucleotide.

At least one modification may be 2′-O-methyl and/or at least onemodification may be 2′-F. Further modifications as described herein maybe present on the first and/or second strand.

Throughout the description of the invention, “same or commonmodification” means the same modification to any nucleotide, be that A,G, C or U modified with a group such as a methyl group or a fluorogroup. Is it not taken to mean the same addition on the same nucleotide.For example, 2′F-dU, 2′F-dA, 2′F-dC, 2′F-dG are all considered to be thesame or common modification, as are 2′-OMe-rU, 2′-OMe-rA; 2′-OMe-rC;2′-OMe-rG. A 2-′F modification is a different modification to a 2′-OMemodification.

Some representative modified nucleic acid sequences of the presentinvention are shown in the examples. These examples are meant to berepresentative and not limiting.

Preferably, the nucleic acid may comprise a modification and a second orfurther modification which are each and individually selected from thegroup comprising 2′-O-methyl modification and 2′-F modification. Thenucleic acid may comprise a modification that is 2′-O-methyl (2′-OMe)that may be a first modification, and a second modification that is2′-F. The nucleic acid of the invention may also include aphosphorothioate modification and/or a deoxy modification which may bepresent in or between the terminal 2 or 3 nucleotides of each or any endof each or both strands.

The invention provides as a further aspect, a nucleic acid forinhibiting expression of LPA in a cell, comprising a nucleotide sequenceof SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, or 43, wherein the nucleotides of first strand aremodified by a first modification on the odd numbered nucleotides, andmodified by a second modification on the even numbered nucleotides, andnucleotides of the second strand are modified by a third modification onthe even numbered nucleotides and modified by a fourth modification theodd numbered nucleotides, wherein at least the first modification isdifferent to the second modification and the third modification isdifferent to the fourth modification. The third and first modificationsmay be the same or different, the second and fourth modifications may bethe same or different. The first and second modifications may bedifferent to each other and the third and fourth modifications may bedifferent to each other.

The second strand may comprise a nucleotide sequence of SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, or 44. The nucleotides of the first strand may be modified by afirst modification on the odd numbered nucleotides, and modified with asecond modification on the even numbered nucleotides, and the secondstrand may be modified on the odd numbered nucleotides with the secondmodification and modified with the first modification on the evennumbered nucleotides. The first modification may be 2′OMe and the secondmodification may be 2′ F. The first strand may comprise the nucleotidesequence of SEQ ID NO: 5 or SEQ ID NO: 9 and/or the second strand maycomprise the nucleotide sequence of SEQ ID NO: 6, or SEQ ID NO:10. Themodifications may be those as set out in Table 1.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotide on the second strand whichcorresponds to position 13 of the first strand is not modified with a 2′O-methyl modification.

A nucleotide on the second strand that “corresponds to” a position onthe first strand is suitably the nucleotide that base pairs with thatnucleotide on the first strand.

In one aspect the nucleotide on the second strand which corresponds toposition 13 of the first strand is the nucleotide that forms a base pairwith position 13 of the first strand.

In one aspect the nucleotide on the second strand which corresponds toposition 11 of the first strand is the nucleotide that forms a base pairwith position 11 of the first strand.

In one aspect the nucleotide on the second strand which corresponds toposition 12 of the first strand is the nucleotide that forms a base pairwith position 12 of the first strand.

This nomenclature may be applied to other positions of the secondstrand. For example, in a 19-mer nucleic acid which is double strandedand blunt ended, position 13 of the first strand would pair withposition 7 of the second strand. Position 11 of the first strand wouldpair with position 9 of the second strand. This nomenclature may beapplied to other positions of the second strand.

The nucleotide that corresponds to position 13 of the first strand issuitably position 13 of the second strand, counting from the 3′ of thesecond strand, starting from the first nucleotide of the double strandedregion. Likewise position 11 of the second strand is suitably the11^(th) nucleotide from the 3′ of the second strand, starting from thefirst nucleotide of the double stranded region. This nomenclature may beapplied to other positions of the second strand.

In one aspect, in the case of a partially complementary first and secondstrand, the nucleotide on the second strand that “corresponds to” aposition on the first strand may not necessarily form a base pair ifthat position is the position in which there is a mismatch, but theprinciple of the nomenclature still applies.

Preferred is a first and second strand that are fully complementary overthe duplex region (ignoring any overhang regions) and there are nomismatches within the double stranded region of the nucleic acid.

Also preferred are:

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotide on the second strand whichcorresponds to position 11 of the first strand is not modified with a 2′O-methyl modification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotides on the second strand whichcorresponds to position 11 and 13 of the first strand are not modifiedwith a 2′ O-methyl modification.

In one aspect the nucleotide on the second strand which corresponds toposition 12 of the first strand is not modified with a 2′ O-methylmodification. This limitation on the nucleic acid may be seen with anyother limitation described herein.

Therefore another aspect of the invention is a nucleic acid as disclosedherein, wherein the nucleotides at positions 2 and 14 from the 5′ end ofthe first strand are not modified with a 2′ O-methyl modification, andthe nucleotides on the second strand which corresponds to position 11-13of the first strand are not modified with a 2′ O-methyl modification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotides on the second strand whichcorrespond to position 11, or 13, or 11 and 13, or 11-13 of the firststrand are modified with a 2′ fluoro modification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are modified with a 2′fluoro modification, and the nucleotides on the second strand whichcorrespond to position 11, or 13, or 11 and 13, or 11-13 of the firststrand are not modified with a 2′ O-methyl modification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are modified with a 2′fluoro modification, and the nucleotides on the second strand whichcorrespond to position 11, or 13, or 11 and 13, or preferably 11-13 ofthe first strand are modified with a 2′ fluoro modification. Preferablyin this embodiment, all even numbered nucleotide of the first strand aremodified with a 2′ fluoro modification and all odd numbered nucleotidesof the first strand are modified with a 2′ O-methyl modification. Inaddition, the nucleotides on the second strand other than those whichcorrespond to position 11, or 13, or 11 and 13, or preferably 11-13 ofthe first strand are modified with a 2′ O-methyl modification. Oneadvantage of such a nucleic is that it comprises relatively fewnon-naturally occurring modified nucleotides but is nonetheless able toefficiently inhibit the target gene for long periods of time. Such anucleic acid is easier to synthesise than corresponding nucleic acidswith more non-naturally occurring (2′F modified) nucleotides.

A nucleic acid as disclosed herein wherein greater than 50% of thenucleotides of the first and/or second strand comprise a 2′ O-methylmodification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%,or more, of the first and/or second strand comprise a 2′ O-methylmodification, preferably measured as a percentage of the totalnucleotides of both the first and second strands.

A nucleic acid as disclosed herein wherein greater than 50% of thenucleotides of the first and/or second strand comprise a naturallyoccurring RNA modification, such as wherein greater than 55%, 60%, 65%,70%, 75%, 80%, or 85% or more of the first and/or second strandscomprise such a modification, preferably measured as a percentage of thetotal nucleotides of both the first and second strands. Suitablenaturally occurring modifications include, as well as 2 O′ methyl, other2′ sugar modifications, in particular a 2′ H modification resulting in aDNA nucleotide.

A nucleic acid as disclosed herein comprising no more than 20%, such asno more than 15% such as no more than 10%, of nucleotides which have 2′modifications that are not 2′ O methyl modifications on the first and/orsecond strand, preferably as a percentage of the total nucleotides ofboth the first and second strands.

A nucleic acid as disclosed herein comprising no more than 20%, (such asno more than 15% or no more than 10%) of 2′ fluoro modifications on thefirst and/or second strand, preferably as a percentage of the totalnucleotides of both strands.

A nucleic acid as disclosed herein, wherein all nucleotides are modifiedwith a 2′ O-methyl modification except positions 2 and 14 from the 5′end of the first strand and the nucleotides on the second strand whichcorrespond to position 11, or 13, or 11 and 13, or preferably 11-13 ofthe first strand. Preferably the nucleotides that are not modified with2′ O-methyl are modified with fluoro at the 2′ position.

Preferred is a nucleic acid as disclosed herein wherein all nucleotidesof the nucleic acid are modified at the 2′ position of the sugar.Preferably these nucleotides are modified with a 2′-fluoro modificationwhere the modification is not a 2′ 0-Methyl modification.

Nucleic acids of the invention may comprise one or more nucleotidesmodified at the 2′ position with a 2′ H, and therefore having a DNAnucleotide within the nucleic acid. Nucleic acids of the invention maycomprise DNA nucleotides at positions 2 and/or 14 of the first strandcounting from the 5′ end of the first strand. Nucleic acids may compriseDNA nucleotides on the second strand which correspond to position 11, or13, or 11 and 13, or 11-13 of the first strand.

In one aspect there is no more than one DNA per nucleic acid of theinvention.

Nucleic acids of the invention may comprise one or more LNA nucleotides.Nucleic acids of the invention may comprise LNA nucleotides at positions2 and/or 14 of the first strand counting from the 5′ end of the firststrand. Nucleic acids may comprise LNA on the second strand whichcorrespond to position 11, or 13, or 11 and 13, or 11-13 of the firststrand.

In one aspect the nucleic acid is modified on the first strand,preferably along the entire strand, with alternating 2′ O-methylmodifications and 2′ fluoro modifications, and positions 2 and 14(starting from the 5′ end) are modified with 2′ fluoro. Preferably thesecond strand is modified with 2′ fluoro modifications at nucleotides onthe second strand which correspond to position 11, or 13, or 11 and 13,or preferably 11-13 of the first strand. Preferably the second strand ismodified with 2′ fluoro modifications at positions 11-13 counting fromthe 3′ end starting at the first position of the complementary (doublestranded) region, and the remaining modifications are naturallyoccurring modifications, preferably 2′ O-methyl. In this case at least,the nucleic acid preferably has a blunt end at least at the end thatcomprises the 5′ end of the first strand.

A nucleotide of the second strand that is in a position correspondingfor example to an even-numbered nucleotide of the first strand is anucleotide of the second strand that is base-paired to an even-numberednucleotide of the first strand.

In one aspect of the nucleic acid, the nucleotide/nucleotides of thesecond strand in a position corresponding to nucleotide 11 or nucleotide13 or nucleotides 11 and 13 or preferably nucleotides 11-13 of the firststrand is/are modified by a fourth modification. Preferably, all thenucleotides of the second strand other than the nucleotide/nucleotidesin a position corresponding to nucleotide 11 or nucleotide 13 ornucleotides 11 and 13 or preferably nucleotides 11-13 of the firststrand is/are modified by a third modification. Preferably in the samenucleic acid nucleotides 2 and 14 or preferably all the even numberednucleotides of the first strand are modified with a first modification.In addition, or alternatively, the odd-numbered nucleotides of the firststrand are modified with a second modification. The fourth modificationis preferably different from the second modification and preferablydifferent from the third modification and the fourth modification ispreferably the same as the first modification. The first and the fourthmodification are preferably a 2′-OMe modification and the second andthird modification are preferably a 2′-F modification. The nucleotideson the first strand are numbered consecutively starting with nucleotidenumber 1 at the 5′ end of the first strand.

In one aspect of the nucleic acid, all the even-numbered nucleotides ofthe first strand are modified by a first modification, all theodd-numbered nucleotides of the first strand are modified by a secondmodification, all the nucleotides of the second strand in positionscorresponding to nucleotides 11-13 of the first strand are modified by afourth modification, all the nucleotides of the second strand other thanthe nucleotides corresponding to nucleotides 11-13 of the first strandare modified by a third modification, wherein the first and fourthmodification are 2′-F and the second and third modification are 2′-OMe.The nucleotides on the first strand are numbered consecutively startingwith nucleotide number 1 at the 5′ end of the first strand.

In one aspect of the nucleic acid, each of the nucleotides of the firststrand and of the second strand is a modified nucleotide.

One aspect is a double-stranded nucleic acid for inhibiting expressionof LPA, preferably in a cell, wherein the nucleic acid comprises a firststrand and a second strand, wherein the first strand sequence comprisesa sequence of at least 15 nucleotides differing by no more than 3nucleotides from any one of the sequences SEQ ID NO: 9, 5, 1, 3, 7, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43,preferably SEQ ID NO: 9, wherein all the even-numbered nucleotides ofthe first strand are modified by a first modification, all theodd-numbered nucleotides of the first strand are modified by a secondmodification, all the nucleotides of the second strand in positionscorresponding to nucleotides 11-13 of the first strand are modified by afourth modification, all the nucleotides of the second strand other thanthe nucleotides corresponding to nucleotides 11-13 of the first strandmodified by a third modification, wherein the first and fourthmodification are 2′-F and the second and third modification are 2′-OMe.

One aspect are nucleic acids which are siRNA molecules wherein thenucleotides at positions 2 and 14 from the 5′ end of the first strandare not modified with a 2′ O-methyl modification, and the nucleic acidcomprises one or more or all of:

-   (i) an inverted nucleotide, preferably a 3′-3′ linkage at the 3′ end    of the second strand;-   (ii) one or more phosphorodithioate linkages;-   (iii) the second strand nucleotide corresponding to position 11 or    13 of the first strand is not modified with a 2′ O-methyl    modification, preferably wherein one or both of these positions    comprise a 2′ fluoro modification;-   (iv) the nucleic acid comprises at least 80% of all nucleotides    having a 2′-O-methyl modification;-   (v) the nucleic acid comprises no more than 20% of nucleotides which    have 2′ fluoro modifications.

Also provided by the present invention is a nucleic acid as disclosedherein, wherein the nucleotides at positions 2 and 14 from the 5′ end ofthe first strand and the nucleotides at positions 7 and/or 9, or 7-9from the 5′ end of the second strand are modified with a 2′ fluoromodification, and at least 90% of the remaining nucleotides are 2′-0methyl modified or comprise another naturally occurring 2′ modification.

Specific preferred examples, for a blunt double stranded 19 base nucleicacid, with no overhang, are:

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotide at position 7 from the 5′ endof the second strand is not modified with a 2′ O-methyl modification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotide at position 9 from the 5′ endof the second strand is not modified with a 2′ O-methyl modification

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotides at position 7 and 9 from the5′ end of the second strand are not modified with a 2′ O-methylmodification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotides at positions 7-9 from the 5′end of the second strand are not modified with a 2′ O-methylmodification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are not modified with a 2′O-methyl modification, and the nucleotides at positions 7 and/or 9, or7-9 from the 5′ end of the second strand are modified with a 2′ fluoromodification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are modified with a 2′fluoro modification, and the nucleotides at positions 7 and/or 9, or 7-9from the 5′ end of the second strand are not modified with a 2′ O-methylmodification.

A nucleic acid as disclosed herein, wherein the nucleotides at positions2 and 14 from the 5′ end of the first strand are modified with a 2′fluoro modification, and the nucleotides at positions 7 and/or 9, or 7-9from the 5′ end of the second strand are modified with a 2′ fluoromodification.

A nucleic acid as disclosed herein wherein greater than 50% of thenucleotides of the first and/or second strand comprise a 2′ O-methylmodification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%,or more, of the first and/or second strand comprise a 2′ O-methylmodification, preferably measured as a percentage of the totalnucleotides of both the first and second strands.

A nucleic acid as disclosed herein wherein greater than 50% of thenucleotides of the first and/or second strand comprise a naturallyoccurring RNA modification, such as wherein greater than 55%, 60%, 65%,70%, 75%, 80%, or 85% or more of the first and/or second strandscomprise such a modification, preferably measured as a percentage of thetotal nucleotides of both the first and second strands. Suitablenaturally occurring modifications include, as well as 2 O′ methyl, other2′ sugar modifications, in particular a 2′ H modification resulting in aDNA nucleotide.

A nucleic acid as disclosed herein comprising no more than 20%, such asno more than 15% such as more than 10%, of nucleotides which have 2′modifications that are not 2′ O methyl modifications on the first and/orsecond strand, preferably as a percentage of the total nucleotides ofboth the first and second strands.

A nucleic acid as disclosed herein comprising no more than 20%, (such asno more than 15% or no more than 10%) of 2′ fluoro modifications on thefirst and/or second strand, preferably as a percentage of the totalnucleotides of both strands.

A nucleic acid as disclosed herein, wherein all nucleotides are modifiedwith a 2′ O-methyl modification except positions 2 and 14 from the 5′end of the first strand and the nucleotides at positions 7 and/or 9 fromthe 5′ end of the second strand. Preferably the nucleotides that are notmodified with 2′ O-methyl are modified with fluoro at the 2′ position.

A nucleic acid as disclosed herein, wherein all nucleotides are modifiedwith a 2′ O-methyl modification except positions 2 and 14 from the 5′end of the first strand and the nucleotides at positions 7-9 from the 5′end of the second strand. Preferably the nucleotides that are notmodified with 2′ O-methyl are modified with fluoro at the 2′ position.

For a nucleic acid comprising a 20 base pair duplex region, the secondstrand preferably does not have a 2′ O-methyl group at nucleotides 8 or9 or 10 counting from the 5′ end of the duplex corresponding topositions 13, 12, and 11 of the first strand respectively.

For a nucleic acid comprising a 21 base pair duplex region, the secondstrand preferably does not have a 2′ O-methyl group at nucleotides 9 or10 or 11 counting from the 5′ end of the duplex corresponding topositions 13, 12, and 11 of the first strand respectively.

The nucleic acid of the present invention may include one or morephosphorothioate modifications on one or more of the ends of the firstand/or the second strand. Optionally, each or either end of the firststrand may comprise one or two or three phosphorothioate modifiednucleotides. Optionally, each or either end of the second strand maycomprise one or two or three phosphorothioate modified nucleotides.

In one embodiment, the first strand may include at least onephosphorothioate (ps) linkage.

In one embodiment, the first strand may further comprise aphosphorothioate linkage between the terminal two 3′ nucleotides orphosphorothioate linkages between the terminal three 3′ nucleotides.

In one embodiment, the linkages between the other nucleotides in thefirst strand are phosphodiester linkages.

In one embodiment, the first strand may include more than 1phosphorothioate linkage.

In a further embodiment, the second strand may comprise aphosphorothioate linkage between the terminal two 3′ nucleotides orphosphorothioate linkages between the terminal three 3′ nucleotides.

In another further embodiment, the second strand may comprise aphosphorothioate linkage between the terminal two 5′ nucleotides orphosphorothioate linkages between the terminal three 5′ nucleotides.

In one aspect the nucleic acid comprises one or more phosphorodithioatelinkages, such as 1, 2, 3 or 4 phosphorodithioate linkages. Preferablythere are up to 4 phosphorodithioate linkages, one each at the 5′ and 3′ends of the first and second strands.

The use of a phosphorodithioate linkage in the nucleic acid of theinvention reduces the variation in the stereochemistry of a populationof nucleic acid molecules compared to molecules comprising aphosphorothioate in that same position. Phosphorothioate linkage indeedintroduce a chiral centre and it is difficult to control whichnon-linking oxygen is substituted for sulphur. The use of aphosphorodithioate ensures that no chiral centre exists in that linkageand thus reduces or eliminates any variation in the population ofnucleic acid molecules, depending on the number of phosphorodithioateand phosphorothioate linkages used in the nucleic acid molecule.

In one aspect, the nucleic acid comprises a phosphorothioate linkagebetween each of the three terminal 3′ nucleotides and/or between each ofthe three terminal 5′ nucleotides on the first strand, and/or betweeneach of the three terminal 3′ nucleotides and/or between each of thethree terminal 5′ nucleotides of the second strand when there is nophosphorodithioate linkage present at that end. No phosphorodithioatelinkage being present at an end means that the linkage between the twoterminal nucleotides, or preferably between the three terminalnucleotides of the nucleic acid end in question are linkages other thanphosphorodithioate linkages.

The invention also provides a nucleic acid according to any aspect ofthe invention described herein, wherein the first RNA strand has aterminal 5′ (E)-vinylphosphonate nucleotide, and the terminal 5′(E)-vinylphosphonate nucleotide is linked to the second nucleotide inthe first strand by a phosphodiester linkage. The first strand mayinclude more than one phosphodiester linkage. In one embodiment, thefirst strand may comprise phosphodiester linkages between at least theterminal three 5′ nucleotides. In one embodiment, the first strand maycomprise phosphodiester linkages between at least the terminal four 5′nucleotides.

In one embodiment, the first strand may comprise formula (IV):(vp)-N(po)[N(po)]_(n)-  (IV)where ‘(vp)-’ is the 5′ (E)-vinylphosphonate, ‘N’ is a nucleotide, ‘po’is a phosphodiester linkage, and n is from 1 to (the total number ofnucleotides in the first strand−2), preferably wherein n is from 1 to(the total number of nucleotides in the first strand−3), more preferablywherein n is from 1 to (the total number of nucleotides in the firststrand−4).

In one aspect, if the 5′-most nucleotide of the first strand is anucleotide other than A or U, this nucleotide is replaced by A or U inthe sequence. Preferably, if the 5′-most nucleotide of the first strandis a nucleotide other than U, this nucleotide is replaced by U, and morepreferably by U with a 5′ vinylphosphonate, in the sequence.

A terminal 5′ (E)-vinylphosphonate nucleotide is a nucleotide whereinthe natural phosphate group at the 5′-end has been replaced with aE-vinylphosphonate, in which the bridging 5′-oxygen atom of the terminalnucleotide of the 5′ phosphorylated strand is replaced with a methynyl(−CH═) group:

5′ (E) vinylphosphonate is a 5′ phosphate mimic. A biological mimic is amolecule that is capable of carrying out the same function as and isstructurally very similar to the original molecule that is beingmimicked. In the context of the present invention, 5′ (E)vinylphosphonate mimics the function of a normal 5′ phosphate, e.g.enabling efficient RISC loading. In addition, because of its slightlyaltered structure, 5′ (E) vinylphosphonate is capable of stabilizing the5′-end nucleotide by protecting it from dephosphorylation by enzymessuch as phosphatases.

In an embodiment, the terminal 5′ (E)-vinylphosphonate nucleotide is anRNA nucleotide.

In one aspect, the nucleic acid:

-   (i) has a phosphorothioate linkage between the terminal three 3′    nucleotides and the terminal three 5′ nucleotides of the first    strand;-   (ii) is conjugated to a triantennary ligand either on the 3′ end    nucleotide or on the 5′ end nucleotide of the second strand;-   (iii) has a phosphorothioate linkage between the terminal three    nucleotides of the second strand at the end opposite to the one    conjugated to the triantennary ligand; and-   (iv) all remaining linkages between nucleotides of the first and/or    of the second strand are phosphodiester linkages.

In one aspect, the nucleic acid:

-   (i) has a terminal 5′ (E)-vinylphosphonate nucleotide at the 5′ end    of the first strand;-   (ii) has a phosphorothioate linkage between the terminal three 3′    nucleotides on the first and second strand and between the terminal    three 5′ nucleotides on the second strand; and-   (iii) all remaining linkages between nucleotides of the first and/or    of the second strand are phosphodiester linkages.

In one aspect, the nucleic acid, which is preferably an siRNA thatinhibits expression of LPA, preferably via RNAi, comprises one or moreor all of:

-   (i) a modified nucleotide;-   (ii) a modified nucleotide other than a 2′-OMe modified nucleotide    at positions 2 and 14 from the 5′ end of the first strand,    preferably a 2′-F modified nucleotide;-   (iii) each of the odd-numbered nucleotides of the first strand as    numbered starting from one at the 5′ end of the first strand are    2′-OMe modified nucleotides;-   (iv) each of the even-numbered nucleotides of the first strand as    numbered starting from one at the 5′ end of the first strand are    2′-F modified nucleotides;-   (v) the second strand nucleotide corresponding to position 11 or 13    of the first strand is modified by a modification other than a    2′-OMe modification, preferably wherein one or both of these    positions comprise a 2′-F modification;-   (vi) an inverted nucleotide, preferably a 3′-3′ linkage at the 3′    end of the second strand;-   (vii) one or more phosphorothioate linkages;-   (viii) one or more phosphorodithioate linkages; and/or-   (ix) the first strand has a terminal 5′ (E)-vinylphosphonate    nucleotide at its 5′ end, in which case the terminal 5′    (E)-vinylphosphonate nucleotide is preferably a uridine and is    preferably linked to the second nucleotide in the first strand by a    phosphodiester linkage.

The nucleic acids of the invention may include one or more invertednucleotides, for example inverted thymidine or inverted adenine (forexample see Takei, et al., 2002. JBC 277 (26):23800-06).

In one aspect the nucleic acid of the invention comprises one or moreinverted ribonucleotides, preferably an inverted adenine, using a 5′-5′linkage or a 3′-3′ linkage, preferably a 3′-3′ linkage at the 3′ end ofthe second strand.

The nucleic acid of the invention may comprise an inverted RNAnucleotide at one or several of the strand ends. Such invertednucleotides provide stability to the nucleic acid. Preferably, thenucleic acid comprises at least an inverted nucleotide at one or severalof the 3′ end of at least one of the strands and/or at the 5′ end of theof the second strand. More preferably, the nucleic acid comprises aninverted nucleotide at the 3′ end of the second strand. Most preferably,the nucleic acid comprises an inverted RNA nucleotide at the 3′ end ofthe second strand and this nucleotide is preferably an inverted A. Theinverted nucleotide is preferably present at an end of a strand not asan overhang but opposite a corresponding nucleotide in the other strand.A nucleic acid with such a modification is stable and easy tosynthesise.

Ligands

The nucleic acid of the invention may be conjugated to a ligand.Efficient delivery of oligonucleotides, in particular double strandednucleic acids of the invention, to cells in vivo is important andrequires specific targeting and substantial protection from theextracellular environment, particularly serum proteins. One method ofachieving specific targeting is to conjugate a ligand to the nucleicacid. The ligand helps in targeting the nucleic acid to the requiredtarget site. There is a need to conjugate appropriate ligands for thedesired receptor molecules in order for the conjugated molecules to betaken up by the target cells by mechanisms such as differentreceptor-mediated endocytosis pathways or functionally analogousprocesses.

One example is the asialoglycoprotein receptor complex (ASGP-R) composedby varying ratios of multimers of membrane ASGR1 and ASGR2 receptors,which is highly abundant on hepatocytes and has high affinity to thehere described GalNAc moiety. One of the first disclosures of the use oftriantennary cluster glycosides as conjugated ligands was in U.S. Pat.No. 5,885,968. Conjugates having three GalNAc ligands and comprisingphosphate groups are known and are described in Dubber et al.(Bioconjug. Chem. 2003 January-February; 14(1):239-46.). The ASGP-Rcomplex shows a 50-fold higher affinity for N-Acetyl-D-Galactosylamine(GalNAc) than D-Gal.

The asialoglycoprotein receptor complex (ASGP-R), which recognizesspecifically terminal β-galactosyl subunits of glycosylated proteins orother oligosaccharides (Weigel, P. H. et. al., Biochim. Biophys. Acta.2002 Sep. 19; 1572(2-3):341-63) can be used for delivering a drug to theliver's hepatocytes expressing the receptor complex by covalent couplingof galactose or galactosamine to the drug substance (Ishibashi, S.; et.al., J Biol. Chem. 1994 Nov. 11; 269(45):27803-6). Furthermore thebinding affinity can be significantly increased by the multi-valencyeffect, which is achieved by the repetition of the targeting moiety(Biessen E A, et al., J Med Chem. 1995 Apr. 28; 38(9):1538-46).

The ASGP-R complex is a mediator for an active uptake of terminalβ-galactosyl containing glycoproteins to the cell's endosomes. Thus, theASGPR is highly suitable for targeted delivery of drug candidatesconjugated to such ligands like, e.g., nucleic acids intoreceptor-expressing cells (Akinc et al., Mol Ther. 2010 July;18(7):1357-64).

More generally the ligand can comprise a saccharide that is selected tohave an affinity for at least one type of receptor on a target cell. Inparticular, the receptor is on the surface of a mammalian liver cell,for example, the hepatic asialoglycoprotein receptor complex describedbefore (ASGP-R).

The saccharide may be selected from N-acetyl galactosamine, mannose,galactose, glucose, glucosamine and fucose. The saccharide may beN-acetyl galactosamine (GalNAc).

A ligand for use in the present invention may therefore comprise (i) oneor more N-acetyl galactosamine (GalNAc) moieties and derivativesthereof, and (ii) a linker, wherein the linker conjugates the GalNAcmoieties to a sequence as defined in any preceding aspects. The linkermay be a bivalent or trivalent or tetravalent branched structure. Thenucleotides may be modified as defined herein.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonlyreferred to in the literature as N-acetyl galactosamine. Reference to“GalNAc” or “N-acetyl galactosamine” includes both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and the α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose. Both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably.Preferably, the compounds of the invention comprise the β-form,2-(Acetylamino)-2-deoxy-β-D-galactopyranose.

The ligand may therefore comprise GalNAc.

The ligand may comprise a compound of formula (I):[S—X¹—P—X²]₃-A-X³—  (I)wherein:

-   -   S represents a saccharide, wherein the saccharide is N-acetyl        galactosamine;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is alkylene or an alkylene ether of the formula        (—CH₂)_(n)—O—CH₂— where n=1-6;    -   A is a branching unit;    -   X³ represents a bridging unit;    -   wherein a nucleic acid according to the present invention is        conjugated to X³ via a phosphate or modified phosphate        (preferably a thiophosphate).

In formula (I), branching unit “A” branches into three in order toaccommodate the three saccharide ligands. The branching unit iscovalently attached to the remaining tethered portions of the ligand andthe nucleic acid. The branching unit may comprise a branched aliphaticgroup comprising groups selected from alkyl, amide, disulphide,polyethylene glycol, ether, thioether and hydroxyamino groups. Thebranching unit may comprise groups selected from alkyl and ether groups.

The branching unit A may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; and

each n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; and

each n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein A₁ is O, S, C═O or NH; and

each n independently represents an integer from 1 to 20.

The branching unit may have the structure:

The branching unit may have the structure:

The branching unit may have the structure:

Optionally, the branching unit consists of only a carbon atom.

The “X³” portion is a bridging unit. The bridging unit is linear and iscovalently bound to the branching unit and the nucleic acid.

X³ may be selected from —C₁-C₂₀ alkylene-, —C₂-C₂₀ alkenylene-, analkylene ether of formula —(C₁-C₂₀ alkylene)-O—(C₁-C₂₀ alkylene)-,—C(O)—C₁-C₂₀ alkylene-, —C₀-C₄ alkylene(Cy)C₀-C₄ alkylene- wherein Cyrepresents a substituted or unsubstituted 5 or 6 membered cycloalkylene,arylene, heterocyclylene or heteroarylene ring, —C₁-C₄alkylene-NHC(O)—C₁-C₄ alkylene-, —C₁-C₄ alkylene-C(O)NH—C₁-C₄ alkylene-,—C₁-C₄ alkylene-SC(O)—C₁-C₄ alkylene-, —C₁-C₄ alkylene-C(O)S—C₁-C₄alkylene-, —C₁-C₄ alkylene-OC(O)—C₁-C₄ alkylene-, —C₁-C₄alkylene-C(O)O—C₁-C₄ alkylene-, and —C₁-C₆ alkylene-S—S—C₁-C₆ alkylene-.

X³ may be an alkylene ether of formula —(C₁-C₂₀ alkylene)-O—(C₁-C₂₀alkylene)-. X³ may be an alkylene ether of formula —(C₁-C₂₀alkylene)-O—(C₄-C₂₀ alkylene)-, wherein said (C₄-C₂₀ alkylene) is linkedto Z. X³ may be selected from the group consisting of —CH₂—O—C₃H₆—,—CH₂—O—C₄H₈—, —CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆—, especially —CH₂—O—C₄H₈—,—CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆—, wherein in each case the —CH₂— group islinked to A.

The ligand may comprise a compound of formula (II):[S—X¹—P—X²]₃-A-X³—  (II)

wherein:

-   -   S represents a saccharide;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is C₁-C₈ alkylene;    -   A is a branching unit selected from:

-   -   X³ is a bridging unit;    -   wherein a nucleic acid according to the present invention is        conjugated to X³ via a    -   phosphate or modified phosphate (preferably a thiophosphate)

Branching unit A may have the structure:

Branching unit A may have the structure:

wherein X³ is attached to the nitrogen atom.

X³ may be C₁-C₂₀ alkylene. Preferably, X³ is selected from the groupconsisting of —C₃H₆—, —C₄H₈—, —C₆H₁₂— and —C₈H₁₆—, especially —C₄H₈—,—C₆H₁₂— and —C₈H₁₆—.

The ligand may comprise a compound of formula (III):[S—X¹—P—X²]₃-A-X³  (III)

wherein:

-   -   S represents a saccharide;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is an alkylene ether of formula —C₃H₆—O—CH₂—;    -   A is a branching unit;    -   X³ is an alkylene ether of formula selected from the group        consisting of —CH₂—O—CH₂—, —CH₂—O—C₂H₄—, —CH₂—O—C₃H₆—,        —CH₂—O—C₄H₈—, —CH₂—O—C₅H₁₀—, —CH₂—O—C₆H₁₂—, —CH₂—O—C₇H₁₄—, and        —CH₂—O—C₈H₁₆—, wherein in each case the —CH₂— group is linked to        A,    -   and wherein X³ is conjugated to a nucleic acid according to the        present invention by a phosphate or modified phosphate        (preferably a thiophosphate).

The branching unit may comprise carbon. Preferably, the branching unitis carbon.

X³ may be selected from the group consisting of —CH₂—O—C₄H₈—,—CH₂—O—C₅H₁₀—, —CH₂—O—C₆H₁₂—, —CH₂—O—C₇H₁₄—, and —CH₂—O—C₈H₁₆—.Preferably, X³ is selected from the group consisting of —CH₂—O—C₄H₈—,—CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆.

For any of the above aspects, when P represents a modified phosphategroup, P can be represented by:

wherein Y¹ and Y² each independently represent ═O, ═S, —O⁻, —OH, —SH,—BH₃, —OCH₂CO₂, —OCH₂CO₂R^(x), —OCH₂C(S)OR^(x), and —OR^(x), whereinR^(x) represents C₁-C₆ alkyl and wherein

indicates attachment to the remainder of the compound.

By modified phosphate it is meant a phosphate group wherein one or moreof the non-linking oxygens is replaced. Examples of modified phosphategroups include phosphorothioate, phosphoroselenates, borano phosphates,borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkylor aryl phosphonates and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulphur. One, each or both non-linkingoxygens in the phosphate group can be independently any one of S, Se, B,C, H, N, or OR (R is alkyl or aryl).

The phosphate can also be modified by replacement of a linking oxygenwith nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at a terminal oxygen. Replacement of thenon-linking oxygens with nitrogen is possible.

For example, Y¹ may represent —OH and Y² may represent ═O or ═S; or

Y¹ may represent —O⁻ and Y² may represent ═O or ═S;

Y¹ may represent ═O and Y² may represent —CH₃, —SH, —OR^(x), or —BH₃

Y¹ may represent ═S and Y² may represent —CH₃, OR^(x) or —SH.

It will be understood by the skilled person that in certain instancesthere will be delocalisation between Y¹ and Y².

Preferably, the modified phosphate group is a thiophosphate group.Thiophosphate groups include bithiophosphate (i.e. where Y¹ represents═S and Y² represents —S⁻) and monothiophosphate (i.e. where Y¹represents —O⁻ and Y² represents ═S, or where Y¹ represents ═O and Y²represents —S⁻). Preferably, P is a monothiophosphate. The inventorshave found that conjugates having thiophosphate groups in replacement ofphosphate groups have improved potency and duration of action in vivo.

P may also be an ethylphosphate (i.e. where Y¹ represents ═O and Y²represents OCH₂CH₃).

The saccharide may be selected to have an affinity for at least one typeof receptor on a target cell. In particular, the receptor is on thesurface of a mammalian liver cell, for example, the hepaticasialoglycoprotein receptor complex (ASGP-R).

For any of the above aspects, the saccharide may be selected fromN-acetyl with one or more of galactosamine, mannose, galactose, glucose,glucosamine and fructose. Typically a ligand to be used in the presentinvention may include N-acetyl galactosamine (GalNAc). Preferably thecompounds of the invention may have 3 ligands, which will eachpreferably include N-acetyl galactosamine.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonlyreferred to in the literature as N-acetyl galactosamine. Reference to“GalNAc” or “N-acetyl galactosamine” includes both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and the α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments,both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably.Preferably, the compounds of the invention comprise the β-form,2-(Acetylamino)-2-deoxy-β-D-galactopyranose.

For any of the above compounds of formula (III), X¹ may be(—CH₂—CH₂—O)(—CH₂)₂—. X¹ may be (—CH₂—CH₂—O)₂(—CH₂)₂—. X¹ may be(—CH₂—CH₂—O)₃(—CH₂)₂—. Preferably, X¹ is (—CH₂—CH₂—O)₂(—CH₂)₂—.Alternatively, X¹ represents C₃-C₆ alkylene. X¹ may be propylene. X¹ maybe butylene. X¹ may be pentylene. X¹ may be hexylene. Preferably thealkyl is a linear alkylene. In particular, X¹ may be butylene.

For compounds of formula (III), X² represents an alkylene ether offormula —C₃H₆—O—CH₂—i.e. C₃ alkoxy methylene, or —CH₂CH₂CH₂OCH₂—.

The invention provides a conjugated nucleic acid having one of thefollowing structures:

wherein Z is a nucleic acid as defined herein before and is preferablyconjugated to the 5′ end of the second strand of the nucleic acid.

Preferably, the conjugated nucleic acid has the following structure:

wherein Z is a nucleic acid as defined herein before and is preferablyconjugated to the 5′ end of the second strand.

A ligand of formula (I), (II) or (III) can be attached at the 3′-end ofthe first (antisense) strand and/or at any of the 3′- and/or 5′-end ofthe second (sense) strand. The nucleic acid can comprise more than oneligand of formula (I), (II), or (III). However, a single ligand offormula (I), (II) or (III) is preferred because a single such ligand issufficient for efficient targeting of the nucleic acid to the targetcells. Preferably in that case, at least the last two, preferably atleast the last three and more preferably at least the last fournucleotides at the end of the nucleic acid to which the ligand isattached are linked by a phosphodiester linkage.

Preferably, the 5′-end of the first (antisense) strand is not attachedto a ligand of formula (I), (II) or (III), since a ligand in thisposition can potentially interfere with the biological activity of thenucleic acid.

A nucleic acid with a single ligand of formula (I), (II) or (III) at the5′-end of a strand is easier and therefore cheaper to synthesis than thesame nucleic acid with the same ligand at the 3′-end. Preferablytherefore, a single ligand of any of formulae (I), (II) or (III) iscovalently attached to (conjugated with) the 5′-end of the second strandof the nucleic acid.

In one embodiment, the nucleic acid is conjugated to a ligand thatcomprises a lipid, and more preferably a ligand that comprises acholesterol.

Alternatively, a nucleic acid according to the present invention may beconjugated to a ligand of the following structure

A conjugate of the invention can comprise any nucleic acid as disclosedherein conjugated to any ligand or ligands as disclosed herein.

The present invention also relates to a conjugate for inhibitingexpression of a LPA gene in a cell, said conjugate comprising a nucleicacid portion, comprising the nucleic acid of any aspect of theinvention, and at least one ligand portion, said nucleic acid portioncomprising at least one duplex region that comprises at least a portionof a first RNA strand and at least a portion of a second RNA strand thatis at least partially complementary to the first strand, wherein saidfirst strand is at least partially complementary to at least a portionof RNA transcribed from said LPA gene, said at least one ligand portioncomprising a linker moiety, preferably a serinol-derived linker moiety,and a targeting ligand for in vivo targeting of cells and beingconjugated exclusively to the 3′ and/or 5′ ends of one or both RNAstrands, wherein the 5′ end of the first RNA strand is not conjugated,wherein:

-   -   (i) the second RNA strand is conjugated at the 5′ end to the        targeting ligand, and wherein (a) the second RNA strand is also        conjugated at the 3′ end to the targeting ligand and the 3′ end        of the first RNA strand is not conjugated; or (b) the first RNA        strand is conjugated at the 3′ end to the targeting ligand and        the 3′ end of the second RNA strand is not conjugated; or (c)        both the second RNA strand and the first RNA strand are also        conjugated at the 3′ ends to the targeting ligand; or    -   (ii) both the second RNA strand and the first RNA strand are        conjugated at the 3′ ends to the targeting ligand and the 5′ end        of the second RNA strand is not conjugated.

The ligands may be monomeric or multimeric (e.g. dimeric, trimeric,etc.).

Suitably, the ligands are monomeric, thus containing a single targetingligand moiety, e.g. a single GalNAc moiety.

Alternatively, the ligands may be dimeric ligands wherein the ligandportions comprise two linker moieties, such as serinol-derived linkermoieties or non-serinol linker moieties, each linked to a singletargeting ligand moiety.

The ligands may be trimeric ligands wherein the ligand portions comprisethree linker moieties, such as serinol-derived linker moieties ornon-serinol linker moieties, each linked to a single targeting ligandmoiety.

The two or three serinol-derived linker moieties may be linked in seriese.g. as shown below:

wherein n is 1 or 2 and Y is S or O.

Preferably, the ligands are monomeric.

Suitably, the conjugated RNA strands are conjugated to a targetingligand via a linker moiety including a further linker wherein thefurther linker is or comprises a saturated, unbranched or branched C₁₋₁₅alkyl chain, wherein optionally one or more carbons (for example 1, 2 or3 carbons, suitably 1 or 2, in particular 1) is/are replaced by aheteroatom selected from O, N, S(O)_(p), wherein p is 0, 1 or 2 (forexample a CH₂ group is replaced with O, or with NH, or with S, or withSO₂ or a —CH₃ group at the terminus of the chain or on a branch isreplaced with OH or with NH₂) wherein said chain is optionallysubstituted by one or more oxo groups (for example 1 to 3, such as 1group).

Suitably, the linker moiety is a serinol-derived linker moiety.

The term “serinol-derived linker moiety” means the linker moietycomprises the following structure:

An O atom of said structure typically links to an RNA strand and the Natom typically links to the targeting ligand.

More suitably, the further linker comprises a saturated, unbranchedC₁₋₁₅ alkyl chain wherein one or more carbons (for example 1, 2 or 3carbons, suitably 1 or 2, in particular 1) is/are replaced by an oxygenatom.

More suitably, the further linker comprises a PEG-chain.

More suitably, the further linker comprises a saturated, unbranchedC₁₋₁₅ alkyl chain.

More suitably, the further linker comprises a saturated, unbranched C₁₋₆alkyl chain.

More suitably, the further linker comprises a saturated, unbranched C₄or C₆ alkyl chain, e.g. a C₄ alkyl chain.

In an embodiment of the invention, the first RNA strand is a compound offormula (X):

-   -   wherein b is 0 or 1; and

the second RNA strand is a compound of formula (XI):

wherein:

-   -   c and d are independently 0 or 1;    -   Z₁ and Z₂ are the RNA portions of the first and second RNA        strands respectively;    -   Y is O or S;    -   n is 0, 1, 2 or 3; and    -   L₁ is a linker to which a ligand is attached;

and wherein b+c+d is 2 or 3.

Suitably, the first RNA strand is a compound of formula (XV)

-   -   wherein b is 0 or 1; and

the second RNA strand is a compound of formula (XVI):

-   -   wherein c and d are independently 0 or 1;

wherein:

-   -   Z₁ and Z₂ are the RNA portions of the first and second RNA        strands respectively;    -   Y is O or S;    -   R₁ is H or methyl;    -   n is 0, 1, 2 or 3; and    -   L is the same or different in formulae (XV) and (XVI) and is        selected from the group consisting of:        -   —(CH₂)_(r)—C(O)—, wherein r=2-12;        -   —(CH₂—CH₂—O)_(s)—CH₂—C(O)—, wherein s=1-5;        -   —(CH₂)_(t)—CO—NH—(CH₂)_(t)—NH—C(O)—, wherein t is            independently 1-5;        -   —(CH₂)_(u)—CO—NH—(CH₂)_(u)—C(O)—, wherein u is independently            1-5; and        -   —(CH₂)_(v)—NH—C(O)—, wherein v is 2-12; and    -   wherein the terminal C(O) (if present) is attached to the NH        group;

and wherein b+c+d is 2 or 3.

Suitably, the first RNA strand is a compound of formula (XII):

-   -   wherein b is 0 or 1; and

the second RNA strand is a compound of formula (XIII):

wherein:

-   -   c and d are independently 0 or 1;    -   Z₁ and Z₂ are the RNA portions of the first and second RNA        strands respectively;    -   Y is O or S;    -   n is 0, 1, 2 or 3; and    -   L₂ is the same or different in formulae (XII) and (XIII) and is        the same or different in moieties bracketed by b, c and d, and        is selected from the group consisting of:

and the terminal OH group is absent such that the following moiety isformed:

-   -   wherein    -   F is a saturated branched or unbranched (such as unbranched)        C₁₋₈alkyl (e.g. C₁₋₆ alkyl) chain wherein one of the carbon        atoms is optionally replaced with an oxygen atom provided that        said oxygen atom is separated from another heteroatom (e.g. an O        or N atom) by at least 2 carbon atoms;    -   L is the same or different in formulae (XII) and (XIII) and is        selected from the group consisting of:        -   —(CH₂)_(r)—C(O)—, wherein r=2-12;        -   —(CH₂—CH₂—O)_(s)—CH₂—C(O)—, wherein s=1-5;        -   —(CH₂)_(t)—CO—NH—(CH₂)_(t)—NH—C(O)—, wherein t is            independently 1-5;        -   —(CH₂)_(u)—CO—NH—(CH₂)_(u)—C(O)—, wherein u is independently            1-5; and        -   —(CH₂)_(v)—NH—C(O)—, wherein v is 2-12; and    -   wherein the terminal C(O) (if present) is attached to the NH        group;

and wherein b+c+d is 2 or 3.

In any one of the above formulae where GalNAc is present, the GalNAc maybe substituted for any other targeting ligand, such as those mentionedherein.

Suitably, b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; b is 1,c is 1 and d is 0; or b is 1, c is 1 and d is 1.

More suitably, b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; orb is 1, c is 1 and d is 1.

Most suitably, b is 0, c is 1 and d is 1.

In one embodiment, Y is O. In another embodiment, Y is S.

In one embodiment, R₁ is H or methyl. In one embodiment, R₁ is H. Inanother embodiment, R₁ is methyl.

In one embodiment, n is 0, 1, 2 or 3. Suitably, n is 0.

In one embodiment, L is selected from the group consisting of:

-   -   —(CH₂)_(r)—C(O)—, wherein r=2-12;    -   —(CH₂—CH₂—O)_(s)—CH₂—C(O)—, wherein s=1-5;    -   —(CH₂)_(t)—CO—NH—(CH₂)_(t)—NH—C(O)—, wherein t is independently        1-5;    -   —(CH₂)_(u)—CO—NH—(CH₂)_(u)—C(O)—, wherein u is independently        1-5; and    -   —(CH₂)_(v)—NH—C(O)—, wherein v is 2-12;    -   wherein the terminal C(O) is attached to the NH group.

Suitably, L is —(CH₂)_(r)—C(O)—, wherein r=2-12. Suitably, r=2-6. Moresuitably, r=4 or 6 e.g. 4.

Suitably, L is:

Example F moieties include (CH₂)₁₋₆ e.g. (CH₂)₁₋₄ e.g. CH₂, (CH₂)₄,(CH₂)₅ or (CH₂)₆, or CH₂O(CH₂)₂₋₃, e.g. CH₂O(CH₂)CH₃.

Suitably, L₂ is:

Suitably, L₂ is:

Suitably, L₂ is:

Suitably, L₂ is:

Suitably, n is 0 and L₂ is:

and the terminal OH group is absent such that the following moiety isformed:

wherein Y is as defined elsewhere herein.

Within the moiety bracketed by b, c and d, L₂ is typically the same.Between moieties bracketed by b, c and d, L₂ may be the same ordifferent. In an embodiment, L₂ in the moiety bracketed by c is the sameas the L₂ in the moiety bracketed by d. In an embodiment, L₂ in themoiety bracketed by c is not the same as L₂ in the moiety bracketed byd. In an embodiment, the L₂ in the moieties bracketed by b, c and d isthe same, for example when the linker moiety is a serinol-derived linkermoiety.

Serinol derived linker moieties may be based on serinol in anystereochemistry i.e. derived from L-serine isomer, D-serine isomer, aracemic serine or other combination of isomers. In a preferred aspect ofthe invention, the serinol-GalNAc moiety (SerGN) has the followingstereochemistry:

i.e. is based on an (S)-serinol-amidite or (S)-serinol succinate solidsupported building block derived from L-serine isomer.

In one embodiment, the targeted cells are hepatocytes.

One aspect is a nucleic acid for inhibiting expression of LPA in a cell,comprising at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of aRNA transcribed from the LPA gene, wherein said first strand comprises anucleotide sequence selected from the following sequences: SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, or 43, wherein the nucleic acid is conjugated to a ligand. Thesecond strand may comprise a nucleotide sequence of SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,or 44. The nucleotides of the first and/or second strand may bemodified, as herein described.

Preferably, the nucleic acid comprises SEQ ID NO:5 or SEQ ID NO:9 andSEQ ID NO:6 or SEQ ID NO:10 conjugated to a ligand of formula (I) (asset out above), wherein the ligand is conjugated to the nucleic acid asdescribed and wherein the first strand is modified with a 2′OMemodification on the odd numbered nucleotides, and modified with a 2′F onthe even numbered nucleotides, and the second strand is modified with a2′OMe on the even numbered nucleotides and modified with a 2′F on theodd numbered nucleotides.

Particularly preferred is a nucleic acid wherein the first strandcomprises, or preferably consists of SEQ ID NO: 165 and the secondstrand optionally comprises, or preferably consists of SEQ ID NO: 163.This nucleic acid can be further conjugated to a ligand. Even morepreferred is a nucleic acid wherein the first strand comprises, orpreferably consists of SEQ ID NO: 165 and the second strand optionallycomprises, or preferably consists of SEQ ID NO: 164. Most preferred isan siRNA that consists of SEQ ID NO: 165 and SEQ ID NO: 164. One aspectof the invention is conjugate 21.

Compositions, Uses and Methods

The present invention also provides pharmaceutical compositionscomprising the nucleic acid or conjugated nucleic acid of the invention.The pharmaceutical compositions may be used as medicaments or asdiagnostic agents, alone or in combination with other agents. Forexample, one or more nucleic acid conjugates of the invention can becombined with a delivery vehicle (e.g., liposomes) and/or excipients,such as carriers, diluents. Other agents such as preservatives andstabilizers can also be added. Methods for the delivery of nucleic acidsare known in the art and within the knowledge of the person skilled inthe art.

The invention also includes a pharmaceutical composition comprising oneor more nucleic acids or conjugated nucleic acids according to thepresent invention in a physiologically/pharmaceutically acceptableexcipient, such as a stabilizer, preservative, diluent, buffer, and thelike.

The pharmaceutical composition may be a sterile injectable aqueoussuspension or solution, or in a lyophilised form or adhered, absorbed orincluded to or into any other suitable galenic carrier substance such aspellets, tablets, capsules, nanoparticles, gels, tablets, beads orsimilar structures.

One aspect relates to a double-stranded nucleic acid that is capable ofinhibiting expression LPA, preferably in a cell, for use as amedicament.

A further aspect of the invention relates to a nucleic acid orconjugated nucleic acid of the invention or the pharmaceuticalcomposition comprising the nucleic acid or conjugated nucleic acid ofthe invention for use in the treatment of a disease, disorder orsyndrome, preferably a disease, disorder or syndrome associated withelevated levels of Lp(a)-containing particles. The treatment may be toprevent and/or reduce the risk to suffer from and/or treat stroke,atherosclerosis, thrombosis or cardiovascular diseases such as coronaryheart disease or aortic stenosis and any other disease or pathologyassociated to elevated levels of Lp(a)-containing particles. Thetreatment may be to prevent and/or reduce the risk of suffering fromand/or treat an atherosclerotic cardiovascular disease, anatherosclerotic cerebrovascular disease, hyperlipidaemia, anddyslipidaemia, preferably wherein the disease is associated withelevated levels of Lp(a)-containing particles. The treatment may be toprevent and/or reduce the risk of suffering from and/or treat calcificaortic stenosis, ischaemic stroke, coronary artery disease, peripheralarterial disease, abdominal aortic aneurysm, heart failure secondary toischaemic cardiomyopathy, or familial hypercholesterolaemia, preferablywherein the disease is associated with elevated levels ofLp(a)-containing particles. Preferable, the treatment is to preventand/or reduce the risk of suffering from and/or treat aortic stenosis,such as calcific aortic stenosis, or familial hypercholesterolaemia,preferably in each case when the disease or disorder is associated withelevated levels of Lp(a)-containing particles. A desirable level ofLp(a)-containing particles in serum is generally described as a level ofunder 14 mg/dL. An elevated level of Lp(a)-containing particles is alevel of at least 14, preferably at least 20, more preferably at least30, more preferably at least 40 and most preferably at least 50 mg/dL ofLp(a)-containing particles in the serum of a subject.

The invention includes a pharmaceutical composition comprising one ormore nucleic acids or conjugated nucleic acids according to the presentinvention in a physiologically/pharmaceutically acceptable excipient,such as a stabiliser, preservative, diluent, buffer and the like.

The terms “Lp(a)-containing particles” and “Lp(a) particles” are usedinterchangeably throughout this disclosure.

The pharmaceutical compositions and medicaments of the present inventionmay be administered to a mammalian subject in a pharmaceuticallyeffective dose. The mammal may be selected from a human, a non-humanprimate, a simian or prosimian, a dog, a cat, a horse, cattle, a pig, agoat, a sheep, a mouse, a rat, a hamster, a hedgehog and a guinea pig,or other species of relevance. On this basis, the wording “LPA” or “LPA”as used herein denotes nucleic acid or protein in any of theabove-mentioned species, if expressed therein naturally or artificially,but preferably this wording denotes human nucleic acids or proteins.

The nucleic acid or conjugated nucleic acid of the present invention canalso be administered in combination with other therapeutic compounds,either administrated separately or simultaneously, e.g., as a combinedunit dose. The further therapeutic agent can be selected from the groupcomprising an oligonucleotide, a small molecule, a monoclonal antibody,a polyclonal antibody and a peptide. A molecular conjugation to otherbiologically active molecular entities such as peptides, cellular orartificial ligands or small and large molecules is also possible.

Dosage levels for the medicament and pharmaceutical compositions of theinvention can be determined by those skilled in the art by routineexperimentation. In one embodiment, a unit dose may contain betweenabout 0.01 mg/kg and about 100 mg/kg body weight of nucleic acid orconjugated nucleic acid. Alternatively, the dose can be from 10 mg/kg to25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kgto 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg body weight, or 0.1mg/kg to 1 mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or0.5 mg/kg to 1 mg/kg body weight. Alternatively, the dose can be fromabout 0.5 mg/kg to about 10 mg/kg body weight, or about 0.6 mg/kg toabout 8 mg/kg body weight, or about 0.7 mg/kg to about 5 mg/kg bodyweight, or about 0.8 mg/kg to about 4 mg/kg body weight, or about 0.9mg/kg to about 3.5 mg/kg body weight, or about 1 mg/kg to about 3 mg/kgbody weight, or about 1 mg/kg body weight, or about 3 mg/kg body weight,wherein “about” is a deviation of up to 30%, preferably up to 20%, morepreferably up to 10%, yet more preferably up to 5% and most preferably0% of the indicated value. Dosage levels may also be calculated viaother parameters such as, e.g., body surface area.

The nucleic acid described herein may be capable of inhibiting theexpression of LPA. The nucleic acid described herein may be capable ofpartially inhibiting the expression of LPA. Inhibition may be complete,i.e. 0% compared of the expression level of LPA in the absence of thenucleic acid of the invention. Inhibition of LPA expression may bepartial, i.e. it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95% or intermediate values of LPA expression in the absence ofa nucleic acid of the invention. Inhibition may last 2 weeks, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 11 weeks, 12 weeks,13 weeks, 14 weeks or up to 3 months, when used in a subject, such as ahuman patient. A nucleic acid or conjugated nucleic acid of theinvention, or compositions including the same, may be for use in aregimen comprising treatments once or twice weekly, every week, everytwo weeks, every three weeks, every four weeks, every five weeks, everysix weeks, every seven weeks, or every eight weeks, or in regimens withvarying dosing frequency such as combinations of the before-mentionedintervals. The nucleic acid may be for use subcutaneously, intravenouslyor using any other application routes such as oral, rectal orintraperitoneal, preferably for use subcutaneously.

In cells and/or subjects treated with or receiving the nucleic acid orconjugated nucleic acid of the present invention, the LPA expression maybe inhibited compared to untreated cells and/or subjects by a range from15% up to 100% but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% or intermediate values.The level of inhibition may allow treatment of a disease associated withLPA expression or overexpression, or may serve to further investigatethe functions and physiological roles of the LPA gene product.

A further aspect of the invention relates to a nucleic acid orconjugated nucleic acid of the invention in the manufacture of amedicament for treating a disease, disorder or syndromes, such as thoseas listed above or additional pathologies associated with elevatedlevels of Lp(a), or additional therapeutic approaches where inhibitionof LPA expression is desired.

Also included in the invention is a method of treating or preventing adisease, disorder or syndrome, such as those listed above, comprisingadministration of a pharmaceutical composition comprising a nucleic acidor conjugated nucleic acid as described herein, to an individual in needof treatment (to improve such pathologies). The nucleic acid compositionmay be administered in a regimen comprising treatments twice every week,once every week, every two weeks, every three weeks, every four weeks,every five weeks, every six weeks, every seven weeks, or every eightweeks or at administration intervals of more than once every eight weeksor in regimens with varying dosing frequency such as combinations of thebefore-mentioned intervals. The nucleic acid or conjugated nucleic acidmay be for use subcutaneously or intravenously or other applicationroutes such as oral, rectal or intraperitoneal. Preferably the nucleicacid or conjugated nucleic acid is injected subcutaneously.

The nucleic acid or conjugated nucleic acid of the present invention canbe produced using routine methods in the art including chemicalsynthesis or expressing the nucleic acid either in vitro (e.g., run offtranscription) or in vivo. For example, using solid phase chemicalsynthesis or using a nucleic acid-based expression vector includingviral derivates or partially or completely synthetic expression systems.In one embodiment, the expression vector can be used to produce thenucleic acid of the invention in vitro, within an intermediate hostorganism or cell type, within an intermediate or the final organism orwithin the desired target cell. Methods for the production (synthesis orenzymatic transcription) of the nucleic acid described herein are knownto persons skilled in the art

All the features of the nucleic acids can be combined with all otheraspects of the invention disclosed herein.

The present invention also relates to the unmodified sequences of allmodified sequences disclosed herein.

The invention will now be described with reference to the followingnon-limiting Figures and Examples.

FIGURES

FIG. 1 shows the results of a non-conjugated siRNA molecule screen forinhibition of LPA mRNA expression in human RT-4 cells.

FIGS. 2A and 2B show the dose response of non-conjugated LPA-targetingsiRNA molecules on LPA mRNA expression in human RT-4 cells.

FIG. 3 shows the inhibition of LPA mRNA expression in human andcynomolgus primary hepatocytes by different doses of GalNAc-L1 LPA-1038conjugated siRNA molecules delivered by receptor-mediated uptake.

FIG. 4 shows representative examples of the knockdown of LPA-mRNA byL6-conjugated GalNAc siRNAs indicated in primary human hepatocytesdelivered by receptor-mediated uptake.

FIG. 5 shows the synthesis of A0268, which is a 3′ mono-GalNAcconjugated single stranded oligonucleotide and is the starting materialin the synthesis of Conjugate 1 and Conjugate 3. (ps) denotesphosphorothioate linkage.

FIG. 6 shows the synthesis of A0006 which is a 5′ tri-antennary GalNAcconjugated single stranded oligonucleotide used for the synthesis ofReference Conjugate 4. (ps) denotes phosphorothioate linkage.

FIGS. 7A, 7B, and 7C illustrate the in vitro determination of TTRknockdown. In particular, FIG. 7A shows the in vitro determination ofTTR knockdown by Reference Conjugates (RC) 1 and 3 as well as theuntreated control “UT”; FIG. 7B shows the in vitro determination of TTRknockdown by Reference Conjugates (RC) 2 and 3, as well as the untreatedcontrol “UT”; and FIG. 7C shows the in vitro determination of TTRknockdown by Conjugates 1, 2 and 3, as well as by RC3 and untreatedcontrol “UT”. Reference Conjugates 1 and 2 represent comparatorconjugates. Reference Conjugate 3 represents a non-targeting GalNAcsiRNA and “untreated” (“UT”) represents untreated cells. Both RC3 and UTare negative controls. mRNA levels were normalised against PtenII.

FIG. 8 shows a time course of serum TTR in c57BL/6 mice cohorts of n=4at 7, 14, and 27 days post s.c. treatment with 1 mg/kg—Conjugates 1-3,Reference Conjugates (RC) 1, 2 and 4 and mock treated (PBS) individuals.

FIG. 9 shows oligonucleotide synthesis of 3′ and 5′ GalNAc conjugatedoligonucleotides precursors (such as compound X0385B-prec).

FIG. 10 shows equal dose response of knock down for LPA targeting siRNAwith two single GalNAc units conjugated to the second strand as comparedto a triantennary GalNAc unit at the 5′ second strand in primarycynomolgus hepatocytes.

FIGS. 11A and 11B illustrate the in vitro determination of TTRknockdown. In particular, FIG. 11A shows the in vitro determination ofTTR knockdown by Conjugates 4, 5, 6 and 2 compared to “Luc” (ReferenceConjugate 3) as well as the untreated control “UT”; FIG. 11B shows thein vitro determination of TTR knockdown by Conjugates 7 and 2, comparedto “Luc” (Reference Conjugate 3) as well as the untreated control “UT”.Luc or Reference Conjugate 3 (RC3) represents a non-targeting GalNAcsiRNA and “untreated” (“UT”) represents untreated cells. Both RC3 and UTare negative controls. mRNA level were normalised against PtenII.

FIGS. 12A and 12B illustrate the in vitro determination of TTRknockdown. In particular, FIG. 12A shows the in vitro determination ofTTR knockdown by Conjugates 8, 9, 10, 11 and 2 compared to “Luc”(Reference Conjugate 3) as well as the untreated control “UT”;

FIG. 12B shows the in vitro determination of TTR knockdown by Conjugates12 and 2, compared to “Luc” (Reference Conjugate 3) as well as theuntreated control “UT”. Luc or Reference Conjugate 3 represents anon-targeting GalNAc siRNA and “untreated” (“UT”) represents untreatedcells. Both RC3 and UT are negative controls. mRNA level were normalisedagainst PtenII.

FIG. 13 illustrates the in vitro determination of LPA mRNA knockdown byConjugate 19 compared to controls. Ctr represents a non-targeting GalNAcsiRNA and “untreated” (“UT”) represents untreated cells. Both Ctr and UTare negative controls. mRNA level were normalised against ACTB.

FIG. 14 shows a time course of Aldh2 liver mRNA levels in c57BL/6 micecohorts of n=6 at 14, 28 and 42 days post s.c. treatment with 1mg/kg—Conjugate 15, Reference Conjugate (RC) 6 and mock treated (PBS)individuals. mRNA level were normalised against Pten.

FIG. 15 shows a time course of Aldh2 liver mRNA levels in c57BL/6 micecohorts of n=6 at 14, 28 and 42 days post s.c. treatment with 1mg/kg—Conjugate 16, Reference Conjugate (RC) 7 and mock treated (PBS)individuals. mRNA level were normalised against Pten.

FIG. 16 shows a time course of Tmprss6 liver mRNA levels in c57BL/6 micecohorts of n=6 at 14, 28 and 42 days post s.c. treatment with 1mg/kg—Conjugate 18, Reference Conjugate (RC) 8 and mock treated (PBS)individuals. mRNA level were normalised against Pten.

FIG. 17 shows serum stability of Conjugates 4, 5, 6, 7 and 2, anduntreated control (UT) at 37° C. over 3 days.

FIG. 18 shows serum stability of Conjugates 8, 9, 10, 11, 12 and 2, anduntreated control (UT) at 37° C. over 3 days.

FIG. 19 shows the reduction in LPA mRNA in primary human hepatocytes byConjugate 21.

FIG. 20 shows the reduction in LPA mRNA in primary cynomolgushepatocytes by Conjugate 21.

FIG. 21 shows that Conjugate 21 does not affect the level of APOB geneexpression.

FIG. 22 shows that Conjugate 21 does not affect the level of PLG geneexpression.

FIG. 23 shows a time course of serum Lp(a) inhibition over 29 days incynomolgus with different dosages of conjugate 21.

FIG. 24 shows a dose response curve of conjugate 21 showing reduction ofserum Lp(a) at day 29 in cynomolgus.

EXAMPLES

The numbering referred to in each example is specific for said example.

Example 1

A number of modified and conjugated siRNA molecules used for functionalexamples are shown here.

LPA-1038 derivatives:

GalNAc-LPA-1038-L1

First strand (SEQ ID NO: 119, based on SEQ ID NO 5)

OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeA3′

Second strand (SEQ ID NO: 120, based on SEQ ID NO SEQ ID NO 6)

5′[ST23 (ps)]3 long trebler(ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU3′

GalNAc-LPA-1038-L6

First strand (SEQ ID NO: 121, based on SEQ ID NO 5)

OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeA3′

Second strand (SEQ ID NO: 122, based on SEQ ID NO 6)

5″[ST23 (ps)]3 ST43(ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU3′

FN (N=A, C, G, U) denotes 2′Fluoro, 2′ DeoxyNucleosides

OMeN (N=A, C, G, U) denotes 2′O Methyl Nucleosides (ps) indicates aphosphorothioate linkage

ST23 and ST43 are as below.

A further example are LPA 1041 derivatives:

GalNAc-LPA-1041-L1

First strand (SEQ ID NO: 123, based on SEQ ID NO 9)

5′OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeG3′

Second strand (SEQ ID NO: 124, based on SEQ ID NO 10)

5′[ST23 (ps)]3 long trebler (ps)FC-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU3′

GalNAc-LPA-1041-L6

First strand (SEQ ID NO: 125, based on SEQ ID NO 9)

5′OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeG3′

Second strand (SEQ ID NO: 126, based on SEQ ID NO 10)

5′[ST23 (ps)]3 ST43 (ps)FC-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU3′

FN (N=A, C, G, U) denotes 2′Fluoro, 2′ DeoxyNucleosides

OMeN (N=A, C, G, U) denotes 2′O Methyl Nucleosides

(ps) indicates a phosphorothioate linkage

All oligonucleotides were either obtained from commercialoligonucleotide manufacturers (Biospring, Frankfurt, Germany, orRiboBio, Guangzhou, Guangdong, PRC) or synthesized on an AKTA oligopilotsynthesizer (in house) using standard phosphoramidite chemistry.Commercially available solid support and 2′O-Methyl RNAphosphoramidites, 2′Fluoro DNA phosphoramidites (all standardprotection) and commercially available long trebler phosphoramidite(Glen research) were used. Synthesis was performed using 0.1 M solutionsof the phosphoramidite in dry acetonitrile and benzylthiotetrazole (BTT)was used as activator (0.3M in acetonitrile). All other reagents werecommercially available standard reagents.

Conjugation of the respective GalNac synthon (e.g., ST23, ST41 or ST43)was achieved by coupling of the respective phosphoramidite to the 5″endof the oligochain under standard phosphoramidite coupling conditions.Phosphorothioates were introduced using standard commercially availablethiolation reagents (EDITH, Link technologies).

The single strands were cleaved off the CPG by using methylamine (40%aqueous) and the resulting crude oligonucleotide was purified by Ionexchange chromatography (Resource Q, 6 mL, GE Healthcare) on a AKTA PureHPLC System using a Sodium chloride gradient. Product containingfractions were pooled, desalted on a size exclusion column (Zetadex, EMPBiotech) and lyophilised.

For annealing, equimolar amounts of the respective single strands weredissolved in water and heated to 80° C. for 5 min. After gradual coolingto RT the resulting duplex was lyophilised.

The sequences of the resulting nucleic acids (siRNAs) are set out inTable 1 below.

TABLE 1 Table 1: Non-conjugated nucleic acid sequences tested forinhibition of LPA mRNA expression. Sequences and applied modificationpattern are indicated SEQ ID NO: siRNA ID strand Sequence Modifications1 LPA-1014 first 5′ucguauaacaauaaggggc3′ 5381616272616284847 strand 2second 5′gccccuuauuguuauacga3′ 4737351615451616382 strand 3 LPA-1024first 5′gauaacucuguccauuacc3′ 8252635354537251637 strand 4 second5′gguaauggacagaguuauc3′ 4816254827282815253 strand 5 LPA-1038 first5′auaacucuguccauuacca3′ 6162717181736152736 strand 6 second5′ugguaauggacagaguuau3′ 1845261846364645161 strand 7 LPA-1040 first5′uaacucuguccauuaccgu3′ 5263535453725163745 strand 8 second5′acgguaauggacagaguua3′ 2748162548272828152 strand 9 LPA-1041 first5′auaacucuguccauuaccg3′ 6162717181736152738 strand 10 second5′cgguaauggacagaguuau3′ 3845261846364645161 strand 11 LPA-1055 first5′agaaugugccucgauaacu3′ 6462545473538252635 strand 12 second5′aguuaucgaggcacauucu3′ 2815253828472725171 strand 13 LPA-1057 first5′auaacucuguccaucacca3′ 6162717181736172736 strand 14 second5′uggugauggacagaguuau3′ 1845461846364645161 strand 15 LPA-1058 first5′auaacucuguccaucaccu3′ 6162717181736172735 strand 16 second5′aggugauggacagaguuau3′ 2845461846364645161 strand 17 LPA 1061 first5′uaacucuguccauuaccau3′ 5263535453725163725 strand 18 second5′augguaauggacagaguua3′ 2548162548272828152 strand 19 LPA-1086 first5′augugccuugauaacucug3′ 6181837154616271718 strand 20 second5′cagaguuaucaaggcacau3′ 3646451617264836361 strand 21 LPA-1099 first5′aguuggugcugcuucagaa3′ 6451845471835172826 strand 22 second5′uucugaagcagcaccaacu3′ 1535462836472736271 strand 23 LPA-1102 first5′aauaaggggcugccacagg3′ 6252648483547363648 strand 24 second5′ccuguggcagccccuuauu3′ 3718184728373715251 strand 25 LPA-1116 first5′uaacucuguccaucaccau3′ 5263535453725363725 strand 26 second5′auggugauggacagaguua3′ 2548182548272828152 strand 27 LPA-1127 first5′augagccucgauaacucug3′ 6182837174616271718 strand 28 second5′cagaguuaucgaggcucau3′ 3646451617464835361 strand 29 LPA-1128 first5′aaugagccucgauaacucu3′ 6254647353825263535 strand 30 second5′agaguuaucgaggcucauu3′ 2828152538284717251 strand 31 LPA-1141 first5′aaugcuuccaggacauuuc3′ 6254715372846361517 strand 32 second5′gaaauguccuggaagcauu3′ 4626181735482647251 strand 33 LPA-1151 first5′acagugguggagaaugugc3′ 6364548184646254547 strand 34 second5′gcacauucuccaccacugu3′ 4727251717363727181 strand 35 LPA-1171 first5′guaugugccucgauaacuc3′ 8161818371746162717 strand 36 second5′gaguuaucgaggcacauac3′ 4645161746483636163 strand 37 LPA-1177 first5′ucgauaacucuguccauca3′ 5382526353545372536 strand 38 secondYugauggacagaguuaucga3′ 1825482728281525382 strand 39 LPA-1189 first5′ugucacuggacauuguguc3′ 5453635482725181817 strand 40 second5′gacacaauguccagugaca3′ 4636362545372818272 strand 41 LPA-1244 first5′cugggauccaugguguaac3′ 7184825372548181627 strand 42 second5′guuacaccauggaucccag3′ 4516363725482537364 strand 43 LPA-1248 first5′agaugaccaagcuuggcag3′ 6461827362835184728 strand 44 second5′cugccaagcuuggucaucu3′ 3547362835184536171 strand Table 1: Nucleotidesmodifications are depicted by the following numbers (column 4), 1 =2′F-dU, 2 = 2′F-dA, 3 = 2′F-dC, 4 = 2′F-dG, 5 = 2′-OMe-rU, 6 =2′-OMe-rA, 7 = 2′-OMe-rC, 8 = 2′-OMe-rG.

TABLE 2 Sequences of LPA, APOB,beta-Actin and PTEN qPCR amplicon setsthat were used to measure mRNA levels are shown below. SEQ ID GeneSpecies Sequences NO: LPA: human 5′AAGTG 45 (upper) TCCTTGC GACGTCC 3′LPA: 5′CCTGG 46 (lower) ACTGTGG GGCTTT 3′ LPA: 5′CTGTT 47 (probe)TCTGAAC AAGCACC AACGGAG C 3′ LPA cynomolgus 5′GTGTC 48 (upper) CTCGCAACGTCCA 3′ LPA 5′GACCC 49 (lower) CGGGGCT TTG 3′ LPA 5′TGGCT 50 (probe)GTTTCTG AACAAGC ACCAATG G 3′ APOB human 5′TCATT 51 (upper) CCTTCCCCAAAGAG ACC 3′ APOB 5′CACCT 52 (lower) CCGTTTT GGTGGTA GAG 3′ APOB5′CAAGC 53 (probe) TGCTCAG TGGAGGC AACACAT TA 3′ beta- human 5′GCATG 54Actin GGTCAGA (upper) AGGATTC CTAT 3′ beta- 5′TGTAG 55 Actin AAGGTGT(lower) GGTGCCA GATT 3′ beta- 5′TCGAG 56 Actin CACGGCA (probe) TCGTCACCAA 3′ beta- cynomolgus 5′AAGG 57 Actin CCAACCG (upper) CGAGAAG 3′ beta-5′AGAGG 58 Actin CGTACAG (lower) GGACAGC A 3′ beta- 5′TGAGA 59 ActinCCTTCAA (probe) CACCCCA GCCATGT AC 3′ PPIB human 5′AGATG 60 (upper)TAGGCCG GGTGATC TTT 3′ PPIB 5′GTAGC 61 (lower) CAAATCC TTTCTCT CCTGT 3′PPIB 5′TGTTC 62 (probe) CAAAAAC AGTGGAT AATTTTG TGGCC 3′

Example 2

Screening of non-conjugated siRNA molecules (Table 1) for inhibition ofLPA mRNA expression in human RT-4 cells.

Liposomal transfection complexes were prepared in triplicate at a ratioof 1.5 μl RNAiMax (ThermoFisher)/80 pmol of the indicated siRNAmolecules. The complex was diluted to the indicated concentrations of2.5 nM and 25 nM, respectively (values represented pairwise as light anddarker grey bars). RT4 human urinary bladder transitional cell papillomacells expressing endogenously LPA were seeded at a density of 125.000cells per well in 24-well format on top of previously platedtransfection complexes (reverse transfection) at the indicatedconcentration. 24 hours after transfection total RNA was isolated usingthe Spin Cell Mini Kit 250 (Stratec). LPA mRNA levels were determined byqRT-PCR relative to PPIB mRNA expression in the respective samples ashousekeeping transcript. Values were normalized to the amount of LPAmRNA detected in untreated cells (intraplate). A non-silencing siRNAcompound was transfected as an additional control. Means and SD ofnormalized triplicate values are shown. Results are shown in FIG. 1.

Example 3

Dose response of non-conjugated LPA-targeting siRNA compounds on LPAmRNA expression in human RT-4 cells.

RT4 human urinary bladder transitional cell papilloma cells werereversely transfected as described above (Example 2) and treated at theindicated concentration (range 100 nM to 0.2 nM) with the differentnon-conjugated siRNA compounds (Table 1) as labeled. 24 h posttransfection, total RNA was isolated using the Spin Cell Mini Kit 250(Stratec). LPA mRNA levels were determined by qRT-PCR relative to PPIBmRNA expression in the respective samples as housekeeping transcript.Values were normalized to the amount of LPA mRNA detected in untreatedcells. The bars represent the remaining LPA mRNA expression for eachdata point. Results are shown in FIG. 2.

Example 4

Inhibition of LPA mRNA expression in human and cynomolgus primaryhepatocytes by different doses of GalNAc-L1 LPA-1038 conjugated siRNAmolecule delivered by receptor-mediated uptake.

Primary hepatocytes (ThermoFisher) were plated on collagen-coated96-well plates at densities of 45,000 cells per well (cynomolgus) and30,000 cells per well (human). GalNAc-L1-conjugated LPA-1038 was addedimmediately after plating at the indicated concentrations (nM). 24 hoursafter siRNA treatment total RNA was isolated using the InviTrap RNA cellHTS 96 well kit (Stratec). LPA mRNA levels were determined by qRT-PCRrelative to Actin (cynomolgus) or APOB (human) mRNA levels in therespective samples as housekeeping transcript. Values were normalized toLPA expression in untreated cells. Means and SD of normalized triplicatevalues of remaining LPA mRNA levels are shown as black bars. Resultsshown in FIG. 3.

Example 5

Knockdown of LPA-mRNA in human primary hepatocytes by the differentindicated L6-GalNAc conjugated siRNAs in primary human hepatocytes uponreceptor-mediated delivery.

Primary human hepatocytes (ThermoFisher) were plated on collagen-coated96-well plates at 30,000 cells per well (96 well format).GalNAc-L6-conjugated siRNAs including a non-silencing control were addedimmediately after cell plating at the two indicated concentrations. 24hours after siRNA treatment total RNA was isolated using the InviTrapRNA cell HTS 96 well kit (Stratec). LPA mRNA expression levels weredetermined by qRT-PCR relative to APOB mRNA as housekeeping transcript.Values were normalized to LPA mRNA expression in untreated cells andremaining LPA mRNA levels represented pairwise as bars (100 nM blackbars, 20 nM grey bars). Means and SD of normalized triplicate values areshown in FIG. 4.

Example 6—In Vitro Determination of TTR Knockdown of Various TTR siRNAGalNAc Conjugates

Murine primary hepatocytes were seeded into collagen pre-coated 96 wellplates (Thermo Fisher Scientific, #A1142803) at a cell density of 30,000cells per well and treated with siRNA-conjugates at concentrationsranging from 10 nM to 0.0001 nM. 24 h post treatment cells were lysedand RNA extracted with InviTrap® RNA Cell HTS 96 Kit/C24×96 preps(Stratec #7061300400) according to the manufactures protocol.Transcripts levels of TTR and housekeeping mRNA (PtenII) were quantifiedby TaqMan analysis.

Target gene expression in primary murine hepatocytes 24 h followingtreatment with the conjugates of the invention, Conjugates 1-3, showedthat target gene expression decreases as the dose of the conjugateincreased compared to the negative controls (see “UT” column andReference Conjugate 3), as shown in FIG. 7. This indicates that thefirst strand is binding to the target gene, thus lowering geneexpression. FIG. 7 also shows the target gene expression levels ofReference Conjugates 1 and 2 which act as comparator conjugates. As canbe seen from a comparison between the data presented in FIGS. 7A and 7C,and 7B and 7C, the conjugates of the invention (Conjugates 1-3) decreasethe target gene expression compared to Reference Conjugates 1 and 2. Themost effective conjugate at 0.01 nM appears to be Conjugate 2. The mosteffective conjugate at 0.1 nM, 0.5 nM, 1 nM and 10 nM appears to beConjugate 3.

Example 7—In Vivo Time Course of Serum TTR in Mice

C₅₇BL/6 mice were treated s.c. with 1 mg/kg siRNA-conjugates at day 0.Serum samples were taken at day 7, 14, and 27 by orbital sinus bleedingand stored at −20° C. until analysis. Serum TTR quantification wasperformed with a Mouse Prealbumin ELISA (ALPCO, 41-PALMS/lot 22,2008003B) according to the manufacturers protocol (sample dilution1:8000 or 1:800).

The results of the time course of serum TTR in c57BL/6 mice cohorts ofn=4 at 7, 14, and 27 days post s.c. treatment with 1 mg/kg Conjugates1-3, Reference Conjugates 1, 2 and 4, and mock treated (PBS) individualsis shown in FIG. 8. As indicated by the data in FIG. 8, the conjugatesof the invention are particularly effective at reducing target geneexpression compared to the negative control (PBS) and ReferenceConjugates 1, 2, and in particular to Reference Conjugate 4. Conjugates2 and 3 are also more effective than Reference Conjugates 1, 2 and 4.The most effective conjugate is Conjugate 2. Thus, it may be expectedthat the dosing level of Conjugate 3 would be about three times lower toachieve the same initial knock down and would also result in longerduration of knock down as compared to Reference Conjugate 4.

More specifically, Conjugate 2 resulted in 3-fold lower target proteinlevel in serum at day seven and 4-fold lower target protein level inserum at day 27 compared to Reference Conjugate 4 at equimolar dose inwild type mice. Furthermore, Conjugate 2 resulted in 85% reduction oftarget serum protein level at day 27 after single injection, compared to36% reduction by equimolar amount of Reference Conjugate 4.

Example 8

Equal dose response of knock down for LPA targeting siRNA with twosingle GalNAc units conjugated to the second strand as compared to atriantennary GalNAc unit at the 5′ second strand in primary cynomolgushepatocytes.

The siRNAs are modified with alternating 2′-OMe/2′-F and contain eachtwo phosphorothioate (PS) internucleotide linkages at their 5′ and 3′terminal two internucleotide linkages. In conjugate 19 oneserinol-GalNAc unit each is attached via a PS-bond to the 5′ and 3′ ofthe second strand. In conjugate 20 the two terminal 5′ internucleotidesof the second strand are phosphodiesters and a triantennary GalNAclinker is attached via a PS bond to this end.

Dose response of LPA knockdown in primary cynomolgus hepatocytes wasassessed 24 h post treatment with 100, 20, 4, 0.8, 0.16, 0.032, and0.006 nM siRNA. The reference control is construct 2, the non-targetingcontrol is named Cte. The transcript ct-value for each treatment groupwas normalized to the transcript ct value for the house keeping gen ACTB(Act) and to untreated hepatocytes, named ut (ΔΔct).

Data are shown in FIG. 10

Material & Methods: siRNAs SEQ ID NO: name batch strand sequence 135Conjugate X0373 X0373A mA (ps) fU 19 (ps) mA fA mC fU mC fU mG fU mC fCmA fU mU fA mC (ps) fC (ps) mG 136 X0373B Ser(GN) (ps) fC (ps) mG (ps)fG mU fA mA fU mG fG mA fC mA fG mA fG mU fU (ps) mA (ps) fU (ps)Ser(GN) 135 Ref. STS200 STS2041 mA (ps) fU Conjugate 41L6 A (ps) mA fA 9mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG 137 STS2041 ST23(ps) B ST23 (ps) ST23 (ps) C6XLT (ps) fC mG fG mU fA mA fU mG fG mA fCmA fG mA fG mU fU (ps) mA (ps) fU 138 Reference X0125 X0125A mC (ps) fUConjugate (ps) mU fA 5 (CTR) mC fU mC fU mC fG mC fC mC fA mA fG mC (ps)fG (ps) mA 139 X0125B [(ST23) (ps)]3 (C6XLT) (ps) fU mC fG mC fU mU fGmG fG mC fG mA fG mA fG mU fA (ps) mA (ps) fG Legend mA, mU, mC, mG2′-O-Methyl RNA fA, fU, fC, fG 2′-deoxy-2′-fluoro RNA (ps)phosphorothioate (po) phosphodiester

Primer: SEQ ID NO: LPA fw GTGTCCTCGCAACGTCCA 48 rev GACCCCGGGGCTTTG 49probe BHQ1-TGGCTGTTTCTGAACAAGCACCAATGG- 140 FAM ACTB fwGCATGGGTCAGAAGGATTCCTAT 54 rev TGTAGAAGGTGTGGTGCCAGATT 55 probeBHQ1-TCGAGCACGGCATCGTCACCAA-VIC 141

General Methods

In Vitro Experiments

Primary murine hepatocytes (Thermo Scientific: GIBCO Lot: #MC798) werethawed and cryo-preservation medium exchanged for Williams E mediumsupplemented with 5% FBS, 1 μM dexamethasone, 2 mM GlutaMax, 1%PenStrep, 4 mg/ml human recombinant insulin, 15 mM Hepes. Cell densitywas adjusted to 250000 cells per 1 ml. 100 μl per well of this cellsuspension were seeded into collagen pre-coated 96 well plates. The testarticle was prediluted in the same medium (5 times concentrated) foreach concentration and 25 μl of this prediluted siRNA or medium onlywere added to the cells. Cells were cultured in at 37° C. and 5% CO₂. 24h post treatment the supernatant was discarded, and cells were washed incold PBS and 250 μl RNA-Lysis Buffer S (Stratec) was added. Following 15min incubation at room temperature plates were storage at −80° C. untilRNA isolation according to the manufacturers protocol.

TaqMan Analysis

For mTTR & PTEN MultiPlex TaqMan analysis 10 μl isolated RNA for eachtreatment group were mixed with 10 μl PCR mastermix (TAKYON low Rox)containing 600 nM mTTR-primer, 400 nM ApoB-primer and 200 nM of eachprobe as well as 0.5 units Euroscript II RT polymerase with 0.2 unitsRNAse inhibitor. TaqMan analysis was performed in 384-well plate with a10 min RT step at 48° C., 3 min initial denaturation at 95° C. and 40cycles of 95° C. for 10 sec and 60° C. for 1 min. The primers containtwo of BHQ1, FAM and YY, one at each end of the sequence.

For TMPRSS6 & ApoB MultiPlex TaqMan analysis 10 μl isolated RNA for eachtreatment group were mixed with 10 μl PCR mastermix (TAKYON low Rox)containing 800 nM TMPRSS6 primer, 100 nM ApoB primer and 200 nM ofeither probe as well as 0.5 units Euroscript II RT polymerase with 0.2units RNAse inhibitor. TaqMan analysis was performed in 384-well platewith a 10 min RT step at 48° C., 3 min initial denaturation at 95° C.and 40 cycles of 95° C. for 10 sec and 60° C. for 1 min.

In Vivo Experiments

To compare in vivo potency of different siRNA conjugates 1 mg/kg siRNAdissolved in PBS was administered sub cutaneous in the scapular regionof c57BL/6 mice. Cohorts of n=6 for were treated with siRNA targetingAldh2 or Tmprss6 at day 1 and sacrificed at selected times points posttreatment. Liver samples were snap frozen in liquid nitrogen and storedat −80° C. until extraction RNA with InviTrap Spin Tissue RNA Mini Kit(stratec) according to the manufacturers manual. Following, transcriptlevel of Aldh2, Tmprss6 and Pten were quantified as described above.

Tritosome Stability Assay

To probe for RNAase stability in the endosomal/lysosomal compartment ofhepatic cells in vitro siRNA was incubated for 0 h, 4 h, 24 h or 72 h inSprague Dawley Rat Liver Tritosomes (Tebu-Bio, Cat N.: R0610.LT, lot:1610405, pH: 7.4, 2.827 Units/ml). To mimic the acidified environmentthe Tritosomes were mixed 1:10 with low pH buffer (1.5M acetic acid,1.5M sodium acetate pH 4.75). 30 μl of this acidified Tritosomes.Following 10 μl siRNA (20 μM) were mixed with and incubated for theindicated times at 37° C. Following incubation RNA was isolated with theClarity OTX Starter Kit-Cartriges (Phenomenex Cat No: KSO-8494)according to the manufactures protocol for biological fluids.Lyophilized RNA was reconstituted in 30 μl H₂O, mixed with 4× loadingbuffer and 5 μl were loaded to a 20% TBE-polyacrylamide gelelectrophoresis (PAGE) for separation qualitative semi-quantitativeanalysis. PAGE was run at 120 V for 2 h and RNA visualized byEthidum-bromide staining with subsequent digital imaging with a BioradImaging system.

Example 9—Synthesis of Conjugates

Example compounds were synthesised according to methods described belowand methods known to the person skilled in the art. Assembly of theoligonucleotide chain and linker building blocks was performed by solidphase synthesis applying phosphoramidite methodology. GalNAc conjugationwas achieved by peptide bond formation of a GalNAc-carboxylic acidbuilding block to the prior assembled and purified oligonucleotidehaving the necessary number of amino modified linker building blocksattached.

Oligonucleotide synthesis, deprotection and purification followedstandard procedures that are known in the art.

All Oligonucleotides were synthesized on an AKTA oligopilot synthesizerusing standard phosphoramidite chemistry. Commercially available solidsupport and 2′O-Methyl RNA phosphoramidites, 2″Fluoro, 2″Deoxy RNAphosphoramidites (all standard protection, ChemGenes, LinkTech) andcommercially available 3′-Amino Modifier TFA Amino C-6 lcaa CPG 500 Å(Chemgenes), Fmoc-Amino-DMT C-7 CE phosphoramidite (GlyC3Am), 3′-AminoModifier C-3 lcaa CPG 500 Å (C3Am), Fmoc-Amino-DMT C-3 CEDphosphoramidite (C3Am) and TFA-Amino C-6 CED phosphoramidite (C6Am)(Chemgenes), 3′-Amino-Modifier C7 CPG (C7Am) (Glen Research),Non-nucleosidic TFA amino Phosphoramidite (Pip), Non-nucleosidic TFAamino Solid Support (PipAm) (AM Chemicals) were used. Per-acetylatedgalactose amine 8 is commercially available.

Ancillary reagents were purchased from EMP Biotech. Synthesis wasperformed using a 0.1 M solution of the phosphoramidite in dryacetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3Min acetonitrile). Coupling time was 15 min. A Cap/OX/Cap or Cap/Thio/Capcycle was applied (Cap: Ac₂O/NMI/Lutidine/Acetonitrile, Oxidizer: 0.1MI₂ in pyridine/H₂O). Phosphorothioates were introduced using standardcommercially available thiolation reagent (EDITH, Link technologies).DMT cleavage was achieved by treatment with 3% dichloroacetic acid intoluene. Upon completion of the programmed synthesis cycles adiethylamine (DEA) wash was performed. All oligonucleotides weresynthesized in DMT-off mode.

Attachment of the serinol-derived linker moiety was achieved by use ofeither base-loaded (S)-DMT-Serinol(TFA)-succinate-lcaa-CPG 10 or a(S)-DMT-Serinol(TFA) phosphoramidite 7 (synthesis was performed asdescribed in literature Hoevelmann et al. Chem. Sci., 2016, 7, 128-135).Tri-antennary GalNAc clusters (ST23/C4XLT or ST23/C6XLT) were introducedby successive coupling of the respective trebler amidite derivatives(C4XLT-phos or C6XLT-phos) followed by the GalNAc amidite (ST23-phos).

Attachment of amino modified moieties (non-serinol-derived linkers) wasachieved by use of either the respective commercially available aminomodified building block CPG or amidite.

The single strands were cleaved off the CPG by 40% aq. methylaminetreatment. The resulting crude oligonucleotide was purified by ionexchange chromatography (Resource Q, 6 mL, GE Healthcare) on a AKTA PureHPLC System using a sodium chloride gradient. Product containingfractions were pooled, desalted on a size exclusion column (Zetadex, EMPBiotech) and lyophilised.

Individual single strands were dissolved in a concentration of 60 OD/mLin H₂O. Both individual oligonucleotide solutions were added together ina reaction vessel. For easier reaction monitoring a titration wasperformed. The first strand was added in 25% excess over the secondstrand as determined by UV-absorption at 260 nm. The reaction mixturewas heated to 80° C. for 5 min and then slowly cooled to RT. Doublestrand formation was monitored by ion pairing reverse phase HPLC. Fromthe UV-area of the residual single strand the needed amount of thesecond strand was calculated and added to the reaction mixture. Thereaction was heated to 80° C. again and slowly cooled to RT. Thisprocedure was repeated until less than 10% of residual single strand wasdetected.

Synthesis of Compounds 2-10

Compounds 2 to 5 and (S)-DMT-Serinol(TFA)-phosphoramidite 7 weresynthesised according to literature published methods (Hoevelmann et al.Chem. Sci., 2016, 7, 128-135).

(S)-4-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoicAcid (6)

To a solution of 5 in pyridine was added succinic anhydride, followed byDMAP. The resulting mixture was stirred at room temperature overnight.All starting material was consumed, as judged by TLC. The reaction wasconcentrated. The crude material was chromatographed in silica gel usinga gradient 0% to 5% methanol in DCM (+1% triethylamine) to afford 1.33 gof 6 (yield=38%). m/z (ESI-): 588.2 (100%), (calcd. forC30H29F3NO8⁻[M-H]⁻ 588.6). 1H-NMR: (400 MHz, CDCl₃) δ [ppm]=7.94 (d, 1H,NH), 7.39-7.36 (m, 2H, CHaryl), 7.29-7.25 (m, 7H, CHaryl), 6.82-6.79 (m,4H, CHaryl), 4.51-4.47 (m, 1H), 4.31-4.24 (m, 2H), 3.77 (s, 6H,2×DMTr-OMe), 3.66-3.60 (m, 16H, HNEt₃ ⁺), 3.26-3.25 (m, 2H), 2.97-2.81(m, 20H, NEt₃), 2.50-2.41 (4H, m), 1.48-1.45 (m, 26H, HNEt₃ ⁺),1.24-1.18 (m, 29H, NEt₃).

(S)-DMT-Serinol(TFA)-Succinate-lcaa-CPG (10)

The (S)-DMT-Serinol(TFA)-succinate (159 mg, 270 umol) and HBTU (113 mg,299 umol) were dissolved in CH₃CN (10 mL). Diisopropylethylamine (DIPEA,94 μL, 540 umol) was added to the solution, and the mixture was swirledfor 2 min followed by addition native amino-lcaa-CPG (500 A, 3 g, aminecontent: 136 umol/g). The suspension was gently shaken at roomtemperature on a wrist-action shaker for 16 h then filtered, and washedwith DCM and EtOH. The solid support was dried under vacuum for 2 h. Theunreacted amines on the support were capped by stirring with aceticanhydride/lutidine/N-methylimidazole at room temperature. The washing ofthe support was repeated as above. The solid was dried under vacuum toyield solid support 10 (3 g, 26 umol/g loading).

GalNAc Synthon (9)

Synthesis of the GalNAc synthon 9 was performed as described in Nair etal. J. Am. Chem. Soc., 2014, 136 (49), pp 16958-16961, in 46% yield overtwo steps.

The characterising data matched the published data.

Synthesis of Oligonucleotides

All single stranded oligonucleotides were synthesised according to thereaction conditions described above and in FIGS. 5 and 6, and areoutlined in Tables 3 and 4.

All final single stranded products were analysed by AEX-HPLC to provetheir purity. Purity is given in % FLP (% full length product) which isthe percentage of the UV-area under the assigned product signal in theUV-trace of the AEX-HPLC analysis of the final product. Identity of therespective single stranded products (non-modified, amino-modifiedprecursors or GalNAc conjugated oligonucleotides) was proved by LC-MSanalysis.

TABLE 3 Single stranded un-conjugated oligonucleotides MW % FLP ProductMW (ESI−) (AEX- (11) Name calc. found HPLC) A0002 STS16001A 6943.3 Da6943.0 Da 86.6% A0006 STS16001BL4 8387.5 Da 8387.5 Da 94.1% A0114STS22006A 6143.8 Da 6143.7 Da 94.3% A0115 STS22006BL1 7855.1 Da 7855.1Da 92.8% A0122 STS22009A 6260.9 Da 6260.6 Da 92.8% A0123 STS22009BL17783.0 Da 7782.9 Da 87.1% A0130 STS18001A 6259.9 Da 6259.8 Da 76.5%A0131 STS18001BL4 7813.2 Da 7813.1 Da 74.3% A0220 STS16001B-5′1xNH26982.2 Da 6982.1 Da 95.7% A0237 STS16001A 6943.3 Da 6943.3 Da 95.6%A0244 STS16001BV1 6845.2 Da 6844.9 Da 98.2% A0264 STS16001AV4-3′1xNH27112.4 Da 7112.2 Da 95.4% A0329 STS16001BV6-3′5′1xNH2 7183.3 Da 7183.2Da 88.8% A0560 STS16001A 6943.3 Da 6943.3 Da 96.7% A0541STS16001BV1-3′5′NH2 7151.3 Da 7151.0 Da 85.6% A0547 STS16001BV16-3′5′NH27119.3 Da 7119.1 Da 89.9% A0617 STS16001BV20-3′5′NH2 7087.3 Da 7086.7 Da90.1% A0619 STS16001BV1-3′5′2xNH2 7521.3 Da 7521.3 Da 93.4% A0680STS16001A 6943.3 Da 6942.9 Da 91.2% A0514 STS22006A 6143.8 Da 6143.7 Da94.6% A0516 STS22009BV11-3′5′NH2 6665.0 Da 6664.8 Da 87.0% A0517STS22009BV11-3′5′NH2 6593.0 Da 6593.0 Da 86.0% A0521 STS12009BV1-3′5′NH26437.7 Da 6437.8 Da 91.1% A0303 STS12209BL4 7665.0 Da 7664.9 Da 90.4%A0304 STS12209A 6393.1 Da 6392.9 Da 77.6% A0319 STS22009A 6260.9 Da6260.5 Da 86.9% A0353 STS12009A 6416.1 Da 6416.1 Da 94.1% A0216STS17001A 6178.8 Da 6178.7 Da 87.2% A0217 STS17001BL6 7937.2 Da 7937.2Da 78.3%

5′1×NH2 means refers to the position (5′ end) and number (1×NH2) of freeserinol derived amino groups which are available for conjugation. Forexample, 1×3′NH2 on A0264 means there is free amino group which can bereacted with GalNAc synthon 9 at the 3′ end of the strand A0264.3′5′1×NH2 means there is one serinol-derived free amino group which canbe reacted with GalNAc linker 9 at the 3′ end and the 5′ end of thestrand.

TABLE 4 Sinale stranded oligonucleotides with 5′ and 3′ modifications %FLP MW MW (ESI-) (AEX- Product Name 5'mod 3'mod calc. found HPLC) A0561STS16001BV1-3′5′1 × NH2 C6Am GlyC3Am 7267.5 Da 7267.5 Da 66.7% A0563STS16001BV1-3′5′1 × NH2 C3Am C3Am 7183.4 Da 7183.1 Da 75.1% A0651STS16001BV1-3′5′1 × NH2 C6Am C7Am 7265.6 Da 7265.2 Da 99.6% A0653STS16001BV1-3′5′1 × NH2 GlyC3Am GlyC3Am 7299.5 Da 7299.3 Da 88.1% A0655STS16001BV1-3′5′1 × NH2 PipAm PipAm 7517.7 Da 7517.5 Da 89.8%

Similarly, 3′5′1×NH2 refers to the position (3′ and 5′ end) and number(1×NH2 each) of free amino groups which are available for conjugation.For example, 3′5′1×NH2 on A0561 means there are 2 free amino group (1 atthe 3′ AND 1 at the 5′ end) which can be reacted with GalNAc synthon 9at the 3′ end of the strand A0561.

Synthesis of Certain Conjugates and Reference Conjugates 1-2

Conjugation of the GalNac synthon (9) was achieved by coupling to theserinol-amino function of the respective oligonucleotide strand 11 usinga peptide coupling reagent. Therefore, the respective amino-modifiedprecursor molecule 11 was dissolved in H₂O (500 OD/mL) and DMSO(DMSO/H₂O, 2,1, v/v) was added, followed by DIPEA (2.5% of totalvolume). In a separate reaction vessel pre-activation of theGalN(Ac4)-C₄-acid (9) was performed by reacting 2 eq. (per aminofunction in the amino-modified precursor oligonucleotide 11) of thecarboxylic acid component with 2 eq. of HBTU in presence of 8 eq. DIPEAin DMSO. After 2 min the pre-activated compound 9 was added to thesolution of the respective amino-modified precursor molecule. After 30min the reaction progress was monitored by LCMS or AEX-HPLC. Uponcompletion of the conjugation reaction the crude product wasprecipitated by addition of 10× iPrOH and 0.1×2M NaCl and harvested bycentrifugation and decantation. To set free the acetylated hydroxylgroups in the GalNAc moieties the resulting pellet was dissolved in 40%MeNH2 (1 mL per 500 OD) and after 15 min at RT diluted in H₂O (1:10) andfinally purified again by anion exchange and size exclusionchromatography and lyophilised to yield the final product 12 (Table 5).

TABLE 5 Single stranded GalNAc-conjugated oligonucleotides MW % FLPProduct Starting MW (ESI−) (AEX- (12) Material Name calc. found HPLC)A0241 A0220 STS16001BL20 7285.5 Da 7285.3 Da 91.8% A0268 A0264STS16001AV4L33 7415.7 Da 7415.4 Da 96.9% A0330 A0329 STS16001BV6L427789.8 Da 7789.8 Da 95.5% A0544 A0541 STS16001BV1L75 7757.9 Da 7757.7 Da93.3% A0550 A0547 STS16001BV16L42 7725.9 Da 7725.7 Da 88.5% A0620 A0617STS16001BV20L75 7693.91 Da  7693.2 Da 90.9% A0622 A0619 STS16001BV1L948734.3 Da 8734.6 Da 82.9% A0519 A0516 STS22006BV11L42 7271.7 Da 7271.7Da 90.0% A0520 A0517 STS22009BV11L42 7199.6 Da 7199.7 Da 92.9% A0522A0521 STS12009BV1L42 7044.4 Da 7044.4 Da 96.0% A0603 A0602STS20041BV1L42 7280.7 Da 7280.4 Da 93.4%

Synthesis of Certain Conjugates of the Invention

Conjugation of the GalNac synthon (9) was achieved by coupling to theamino function of the respective oligonucleotide strand 14 using apeptide coupling reagent. Therefore, the respective amino-modifiedprecursor molecule 14 was dissolved in H₂O (500 OD/mL) and DMSO(DMSO/H₂O, 2/1, v/v) was added, followed by DIPEA (2.5% of totalvolume). In a separate reaction vessel pre-activation of theGalN(Ac4)-C₄-acid (9) was performed by reacting 2 eq. (per aminofunction in the amino-modified precursor oligonucleotide 14) of thecarboxylic acid component with 2 eq. of HBTU in presence of 8 eq. DIPEAin DMSO. After 2 min the pre-activated compound 9 was added to thesolution of the respective amino-modified precursor molecule. After 30min the reaction progress was monitored by LCMS or AEX-HPLC. Uponcompletion of the conjugation reaction the crude product wasprecipitated by addition of 10+ iPrOH and 0.1×2M NaCl and harvested bycentrifugation and decantation. To set free the acetylated hydroxylgroups in the GalNAc moieties the resulting pellet was dissolved in 40%MeNH2 (1 mL per 500 OD) and after 15 min at RT diluted in H₂O (1:10) andfinally purified again by anion exchange and size exclusionchromatography and lyophilised to yield the final product 15 (Table 6).

TABLE 6 Single stranded GalNAc-conjugated oligonucleotides MW % FLPProduct Starting MW (ESI−) (AEX- (15) Material Name calc. found HPLC)A0562 A0561 STS16001BV1L87 7874.2 Da 7874.0 Da 82.7% A0564 A0563STS16001BV1L88 7790.0 Da 7789.4 Da 90.4% A0652 A0651 STS16001BV1L967872.2 Da 7871.8 Da 94.6% A0654 A0653 STS16001BV1L97 7906.2 Da 7905.6 Da89.9% A0656 A0655 STS16001BV1L98 8124.3 Da 8124.0 Da 93.6%

Double Strand Formation

Double strand formation was performed according to the methods describedabove.

The double strand purity is given in % double strand which is thepercentage of the UV-area under the assigned product signal in theUV-trace of the IP-RP-HPLC analysis (Table 7).

TABLE 7 Nucleic acid conjugates Starting Materials First Second % doubleProduct Strand Strand Name strand Ref. Conj. 1 A0237 A0241 STS16001L2097.7% Ref. Conj. 2 A0268 A0244 STS16001L33 97.8% Ref. Conj. 3 A0130A0131 STS18001L4 96.8% Ref. Conj. 4 A0002 A0006 STS16001L4 90.1% Ref.Conj. 5 A0216 A0217 STS17001L6 88.4% Conjugate 1 A0268 A0241 STS16001L2496.0% Conjugate 2 A0237 A0330 STS16001V1L42 98.5% Conjugate 3 A0268A0330 STS16001V1L43 98.2% Conjugate 4 A0560 A0544 STS16001V1L75 92.5%Conjugate 5 A0560 A0550 STS16001V16L42 95.3% Conjugate 6 A0237 A0620STS16001V20L75 97.8% Conjugate 7 A0237 A0622 STS16001V1L94 93.7%Conjugate 8 A0680 A0652 STS16001V1L96 98.4% Conjugate 9 A0680 A0654STS16001V1L97 95.8% Conjugate 10 A0680 A0656 STS16001V1L98 97.6%Conjugate 11 A0560 A0564 STS16001V1L88 95.0% Conjugate 12 A0237 A0562STS16001V1L87 96.8% Conjugate 13 A0114 A0115 STS22006L1 85.6% Conjugate14 A0122 A0123 STS22009L1 96.4% Conjugate 15 A0514 A0519 STS22006V11L4298.6% Conjugate 16 A0319 A0520 STS22009V11L42 97.0% Conjugate 17 A0304A0303 STS12209L4 93.0% Conjugate 18 A0353 A0522 STS12009V1L42 98.0%Conjugate 19 A0601 A0603 STS20041BL42 97.6%

Sequences

Modifications key for the following sequences:

f denotes 2′Fluoro 2′deoxyribonucleotide or 2′-fluoro ribonucleotide(the terms are interchangeable)

m denotes 2′O Methyl ribonucleotide

(ps) denotes phosphorothioate linkage

FAM=6-Carboxyfluorescein

BHQ=Black Hole Quencher 1

YY=Yakima Yellow

Definitions

Ser(GN) is a GalNAc-C4 building block attached to serinol derived linkermoiety:

wherein the O— is the linkage between the oxygen atom and e.g. H,phosphordiester linkage or phosphorothioate linkage.

GN is:

C4XLT is:

C6XLT is:

ST23 is:

Synthesis of the phosphoramidite derivatives of C4XLT (C4XLT-phos),C6XLT (C6XLT-phos) as well as ST23 (ST23-phos) can be performed asdescribed in WO2017/174657.

C3Am is:

Itrb is:

GlyC3Am is:

C6Am is:

Pip Am is:

C7Am is:

wherein G = H (pre conjugation) or G = GN (post conjugation).

Conjugate 1

Antisense strand—STS16001AL33 (SEQ ID NO: 127)

5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps) fU (ps) mU (ps) Ser(GN) 3′

Sense strand—STS16001BL20 (SEQ ID NO: 128)

5′ Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU(ps) mA (ps) fA 3′

Conjugate 2

Antisense strand—STS16001A (SEQ ID NO: 129)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU

Sense strand—STS16001BV1L42 (SEQ ID NO: 130)

Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mCfU mA fU (ps) mA (ps) fA (ps) Ser(GN)

Conjugate 3

Antisense strand—STS16001AL33 (SEQ ID NO: 127)

5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps) fU (ps) mU (ps) Ser(GN) 3′

Sense strand—STS16001BV1L42 (SEQ ID NO: 130)

5′ Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fUmC fU mA fU (ps) mA (ps) fA (ps) Ser(GN) 3′

Conjugate 4

Antisense strand—STS16001A (SEQ ID NO: 129)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU

Sense strand—STS16001BV1L75 (SEQ ID NO: 142)

5′ Ser(GN) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fUmA fU (ps) mA (ps) fA Ser(GN) 3′

Conjugate 5

Antisense strand—STS16001A (SEQ ID NO: 129)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU

Sense strand—STS16001BV16L42 (SEQ ID NO: 143)

5′ Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fUmA fA (ps) Ser(GN) 3′

Conjugate 6

Antisense strand—STS16001A (SEQ ID NO: 129)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU

Sense strand—STS16001BV20L75 (SEQ ID NO: 144)

5′ Ser(GN) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU mAfA Ser(GN) 3′

Conjugate 7

Antisense strand—(SEQ ID NO: 129)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU

Sense strand—STS16001BV1L94 (SEQ ID NO: 145)

Ser(GN) (ps) Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mUfG mC fU mC fU mA fU (ps) mA (ps) fA (ps) Ser(GN) (ps) Ser(GN) 3′

Conjugate 8

Antisense strand—STS16001A (SEQ ID NO: 129)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU 3′

Sense strand—STS16001V1BL96 (SEQ ID NO: 146)

5′ C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fUmC fU mA fU (ps) mA (ps) fA (ps) C7Am(GN) 3′

Conjugate 9

Antisense strand—STS16001A (SEQ ID NO: 129)

5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps) fU (ps) mU 3′

Sense strand—STS16001V1BL97 (SEQ ID NO: 147)

5′ GlyC3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mCfU mC fU mA fU (ps) mA (ps) fA (ps) GlyC3Am(GN) 3′

Conjugate 10

Antisense strand—STS16001A (SEQ ID NO: 129)

5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps) fU (ps) mU 3′

Sense strand (SEQ ID NO: 148)

5′ PipAm(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fUmC fU mA fU (ps) mA (ps) fA (ps) PipAm(GN) 3′

Conjugate 11

Antisense strand—STS16001A (SEQ ID NO: 129)

5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps) fU (ps) mU 3′

Sense strand—STS16001V1BL88 (SEQ ID NO: 149)

5′ C3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fUmC fU mA fU (ps) mA (ps) fA (ps) C3Am(GN) 3′

Conjugate 12

Antisense strand—STS16001A (SEQ ID NO: 129)

5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps) fU (ps) mU 3′

Sense strand—STS16001V1BL87 (SEQ ID NO: 150)

5′ C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fUmC fU mA fU (ps) mA (ps) fA (ps) GlyC3Am(GN)

Conjugate 15

Antisense strand (SEQ ID NO: 151)

mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU (ps) fU(ps) mC

Sense strand (SEQ ID NO: 152)

Ser(GN) (ps) fG (ps) mA (ps) fA mA fC mU fC mA fG mU fU mU fA mA fG mAfA (ps) mG (ps) fA (ps) Ser(GN)

Conjugate 16

Antisense strand (SEQ ID NO: 153)

mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU (ps) fC(ps) mU

Sense strand (SEQ ID NO: 154)

Ser(GN) (ps) fA (ps) mG (ps) fA mA fG mA fU mC fC mU fC mG fG mC fU mAfC (ps) mA (ps) fU (ps) Ser(GN)

Conjugate 18

Antisense strand (SEQ ID NO: 155)

mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG(ps) mA

Sense strand (SEQ ID NO: 156)

Ser(GN) (ps) fU (ps) mC (ps) fA mC fC mU fG mC fU mU fC mU fU mC fU mGfG (ps) mU (ps) fU (ps) Ser(GN)

Conjugate 19

Antisense strand (SEQ ID NO: 135)

mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC(ps) mG

Sense strand (SEQ ID NO: 136)

Ser(GN) (ps) fC (ps) mG (ps) fG mU fA mA fU mG fG mA fC mA fG mA fG mUfU (ps) mA (ps) fU (ps) Ser(GN)

Reference Conjugate 1

Antisense strand—STS16001A (SEQ ID NO: 129)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU

Sense strand—STS16001 BL20 (SEQ ID NO: 128)

Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU(ps) mA (ps) fA

Reference Conjugate 2

Antisense strand—STS16001AL33 (SEQ ID NO: 127)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU (ps) Ser(GN)

Sense strand—STS16001BV1 (SEQ ID NO: 157)

fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps)mA (ps) fA

Reference Conjugate 3—“Luc”

Antisense strand—STS18001A (A0130, SEQ ID NO: 132)

mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA (ps) fC(ps) mG Sense strand—STS18001BL4 (A0131, SEQ ID NO: 133)

[(ST23) (ps)]₃ C4XLT (ps) fC mG fU mA fC mG fC mG fG mA fA mU fA mC fUmU fC (ps) mG (ps) fA

Reference Conjugate 4

Antisense strand—STS16001AL33 (SEQ ID NO: 127)

mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps)fU (ps) mU

Sense strand—STS16001BL4 (SEQ ID NO: 134)

5″[(ST23) (ps)]₃ C4XLT(ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mUfG mC fU mC fU mA fU (ps) mA (ps) fA

Reference Conjugate 5—“Ctr”

Antisense strand (SEQ ID NO: 138)

mC (ps) fU (ps) mU fA mC fU mC fU mC fG mC fC mC fA mA fG mC (ps) fG(ps) mA

Sense strand (SEQ ID NO: 139)

[(ST23) (ps)]3 (C6XLT) (ps) fU mC fG mC fU mU fG mG fG mC fG mA fG mA fGmU fA (ps) mA (ps) fG

Reference Conjugate 6

Antisense strand (SEQ ID NO: 151)

mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU (ps) fU(ps) mC

Sense strand (SEQ ID NO: 158)

[ST23 (ps)]3 ltrb (ps) fG mA fA mA fC mU fC mA fG mU fU mU fA mA fG mAfA (ps) mG (ps) fA

Reference Conjugate 7

Antisense strand (SEQ ID NO: 153)

mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU (ps) fC(ps) mU

Sense strand (SEQ ID NO: 159)

[ST23 (ps)]3 ltrb (ps) fA mG fA mA fG mA fU mC fC mU fC mG fG mC fU mAfC (ps) mA (ps) fU

Reference Conjugate 8

Antisense strand (SEQ ID NO: 160)

mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG(ps) mA

Sense strand (SEQ ID NO: 161)

[ST23 (ps)]3 ST41 (ps)fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG(ps) mU (ps) fA

Reference Conjugate 9

Antisense strand (SEQ ID NO: 135)

mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC(ps) mG

Sense strand (SEQ ID NO: 162)

[ST23 (ps)]3 C6XLT (ps) fC mG fG mU fA mA fU mG fG mA fC mA fG mA fG mUfU (ps) mA (ps) fU

Example 10—In Vitro Determination of TTR Knockdown of Various TTR siRNAGalNAc Conjugates

Conjugates 4 to 7

The method described above under “In vitro experiments” in the GeneralMethod section was followed.

Target gene expression in primary murine hepatocytes 24 h followingtreatment at 0.01 nM, 0.1 nM, 0.5 nM, 1 nM and 10 nM with the conjugatesof the invention, Conjugates 4-7, showed that target gene expressiondecreases as the dose of the conjugate increased compared to thenegative controls (see “UT” column and Luc [Reference Conjugate 3]), asshown in FIG. 11. This indicates that the first strand is binding to thetarget gene, thus lowering gene expression.

The in vitro data show that in the context of one or two serinol-derivedlinker moieties being provided at 5′ and 3′ ends of the sense strand inConjugates 4-7, the number of phosphorothioate (PS) bonds between theterminal nucleotide and the linker, and/or between the terminal threenucleotides in the sense strand, can be varied whilst maintainingefficacy for decreasing target gene expression.

Conjugates 8 to 12 and 19

The method described above under “In vitro experiments” in the GeneralMethod section was followed.

Target gene expression in primary murine hepatocytes 24 h followingtreatment at 0.01 nM, 0.1 nM, 0.5 nM, 1 nM and 10 nM with the conjugatesof the invention, Conjugates 8-12, showed that target gene expressiondecreases as the dose of the conjugate increased compared to thenegative controls (see “UT” column and Luc [Reference Conjugate 3]), asshown in FIG. 12. This indicates that the first strand is binding to thetarget gene, thus lowering gene expression. In particular, Conjugates 8,9, 10 and 11 appear to be comparable to or better than Conjugate 2 whichwas previously shown to be the most effective conjugate at 0.01 nM.

Conjugate 19 was also shown to decrease target gene expression comparedto the negative controls (see “UT” column and Ctr which is anon-targeting siRNA and also referred to as Reference Conjugate 5), asshown in FIG. 13. This indicates that the first strand is binding to thetarget gene, thus lowering gene expression.

The in vitro data for Conjugates 8-12 and 19 show that a number oflinkers which are structurally diverse and which are conjugated at bothtermini of the sense strand are effective at decreasing target geneexpression. Conjugates 8-12 and 19 decrease target gene expression moreeffectively than “Luc” which is Reference Conjugate 3 (for Conjugates8-12), “Ctr” which is Reference Conjugate 5 (for Conjugate 19) anduntreated control.

Example 11—In Vivo Time Course of Serum Ttr, Aldh2 and Tmprss6 in Mice

Conjugates 15 to 18

The method described above under “In vivo experiments” in the GeneralMethod section was followed.

The results of the time course of serum Aldh2 in c57BL/6 mice cohorts ofn=6 at 14, 28 and 42 days post s.c. treatment with 1 mg/kg Conjugates 15and 16, Reference Conjugates 6 and 7, and mock treated (PBS) individualsis shown in FIGS. 14 and 15. As indicated by the data in FIGS. 14 and15, the conjugates of the invention are particularly effective atreducing target gene expression compared to the negative control (PBS)and Reference Conjugates 6 and 7 respectively.

The results of the time course of serum Tmprss6 in c57BL/6 mice cohortsof n=6 at 14, 28 and 42 days post s.c. treatment with 1 mg/kg Conjugate18, Reference Conjugate 8, and mock treated (PBS) individuals is shownin FIG. 16. As indicated by the data in FIG. 16, the conjugates of theinvention are particularly effective at reducing target gene expressioncompared to the negative control (PBS) and Reference Conjugate 8.

Overall, the in vivo data show that a variety of example linkers whichare conjugated at both termini of the second strand are effective atdecreasing target gene expression in vivo. The positioning of the linkerimproves in vivo potency conjugates, as compared to a triantennaryGalNAc-linker control at the 5′ terminus of the second strand (ReferenceConjugates 6, 7 and 8).

Example 12—Serum Stability Studies

The method described above under “Tritosome stability assay” in theGeneral Method section was followed.

FIG. 17 shows the results from the serum stability studies in respect ofConjugates 2, 4, 5, 6 and 7. FIG. 18 shows the serum stability ofConjugates 2, 8, 9, 10, 11 and 12.

All conjugates of the invention that were tested are more stable inserum compared to control.

All tested conjugates contain each one GalNAc linker unit at the 5′ endand another at the 3′ end of the second strand. The siRNAs are modifiedwith alternating 2′-OMe/2′-F and contain each two phosphorothioate (PS)internucleotide linkages at their 5′ and 3′ terminal two internucleotidelinkages, unless stated differently.

In Conjugate 4 the serinol-GalNAc units are attached via aphosphodiester bond. In Conjugate 5 the serinol-GalNAc units areconjugated via PS, whereas all internucleotide linkage in the secondstrand are phosphodiesters. In Conjugate 6 the second strand contains noPS. In Conjugate 7 two serinol-GalNAc units are attached to each secondstrand terminus and to each other via a PS-bonds at the respective ends.In Conjugate 8 a C6-amino-modifier at 5′ and a C7-amino-modifier at the3′ end of the second strand were applied for ligand attachment. InConjugate 9 Gly-C₃-amino-modifiers, in Conjugate 10piperidyl-amino-modifiers, in Conjugate 11 C₃-amino-modifiers and inConjugate 2 serinol-GalNAc units were used as linkers for conjugation toboth ends of the second strand. In Conjugate 2 both terminalinternucleotides as well as the nucleotide-serinol bonds are PS. InConjugate 12 a C₆-amino-modifier at the 5′ and a GlyC3-amino-modifier atthe 3′ end of second strand were applied for ligand attachment. “ut”indicates an untreated sample which the other samples were normalisedto. “Luc” indicates an siRNA targeting Luciferase (Reference Conjugate3), which was used as non-targeting control and does not reduce targetmRNA levels.

The data show that in context of a serinol-derived linker moiety beingprovided at 5′ and 3′ ends of the sense strand, the number ofphosphorothioate (PS) bonds between the terminal nucleotide and thelinker, and/or between the terminal three nucleotides in the sensestrand, can be varied whilst maintaining stability in serum.

Example 13

Primary human (Lot Hu1823) and cynomolgus (Lot CY367) hepatocytes andmedia were sourced from Life Technologies. As described by themanufacturer, primary hepatocytes were thawed and plated in platingmedia consisting of Williams' E medium (Life Technologies), supplementedwith 5% fetal bovine serum, 1 μM Dexamethasone in DMSO (finalconcentration of DMSO=0.01%) and 3.6% v/v of Thawing/Plating Cocktail-A(Thermo Fisher Scientific, CM3000).

Human primary hepatocytes were seeded into collagen I-coated 96-wellplates (Life Technologies) at a density of 30,000 cells per well.Cynomolgus hepatocytes were seeded at a density of 45,000 cells perwell. Conjugate 21 and a control GalNAc-conjugated siRNA were seriallydiluted 5-fold at a concentration range of 0.006-100 nM and addedimmediately after plating in plating media. Plates were then incubatedat 37° C. in a 5% CO₂ atmosphere for 24 hours. Subsequently, cells werelysed and RNA was isolated using the method described below.

Conjugate 21 Sequences

Antisense strand—Conjugate 21 (SEQ ID NO: 165)

5′ mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC(ps) mG 3′

Sense strand—STS16001BL20 (SEQ ID NO: 164)

5′ [ST23 (ps)]3 C6XLT (ps) mC mG mG mU mA mA fU fG fG mA mC mA mG mA mGmU mU (ps) mA (ps) mU 3′

Total RNA was extracted using the InviTrap HTS 96-well kit (StratecMolecular GmbH, Berlin, Germany) according to the manufacturer'sinstructions with the following changes to the protocol: After the lastwashing step, plates were centrifuged at 6,000 rpm for twenty minutes.Subsequently, the RNA binding plate was positioned on top of an elutionplate, and RNA was eluted by two rounds of adding 30 μl of elutionbuffer and incubating for two minutes at room temperature, followed byone minute of centrifugation at 1,000 rpm. A final elution step wasperformed by centrifuging at 1700 g (4000 rpm) for 3 minutes RNA wasstored at −80° C.

Ten μl of RNA-solution was used for gene expression analysis by reversetranscription quantitative polymerase chain reaction (RT-qPCR) performedwith amplicon sets/sequences for LPA, ACTB (Eurogentec Deutschland GmbH,Cologne, Germany), APOB and PLG (BioTez GmbH, Berlin, Germany).

The RT-qPCR reactions were carried out with an ABI StepOne Plus (AppliedBiosystems, part of Thermo Fisher Scientific, Massachusetts, USA) usingstandard protocols for RT-PCR (48° C. 30 min, 95° C. 10 min, 40 cyclesat 95° C. 15 s followed by 60° C. 1 min).

In primary hepatocytes, LPA and PLG analyses were performed insingleplex assays (primers: 300 nM, probe: 100 nM). APOB (200 nM) andACTB (300 nM) were run in a multiplex assay adding 100 nM of each probeto the standard mixture.

The data were calculated by using the comparative CT method also knownas the 2^(−ΔΔCt) method (Livak and Schmittgen, 2001 and Schmittgen andLivak, 2008). Here the amount of APOB, LPA or PLG mRNA normalised to theendogenous reference ACTB relative to a calibrator (untreated control)is given by the formulaFold-change=2^(−ΔΔCt).

Unless stated otherwise, all values presented in the example refer tomean±SD. IC₅₀ values were calculated using a sigmoidal 4 parameter doseresponse curve in GraphPad Prism 7.

FIG. 19 shows the knockdown of LPA mRNA by Conjugate 21 throughreceptor-mediated uptake into primary human hepatocytes 24 hours postsiRNA treatment (IC₅₀=3.6 nM). LPA mRNA expression levels werenormalised to ACTB and relative to cells treated with a non-targetingsiRNA control measured by RT-qPCR. Data is presented as the mean±SD of asingle experiment. FIG. 20 shows the knockdown of LPA mRNA by Conjugate21 through receptor-mediated uptake into primary cynomolgus hepatocytes24 hours post siRNA treatment (IC₅₀=0.7 nM). LPA mRNA expression levelswere normalised to ACTB and relative to cells treated with anon-targeting siRNA control measured by RT-qPCR. Data is presented asthe mean±SD of a single experiment.

FIG. 21 shows Knockdown of APOB mRNA by Conjugate 21 throughreceptor-mediated uptake into primary human hepatocytes 24 hours postsiRNA treatment. APOB mRNA expression levels were normalised to ACTB andrelative to cells treated with a non-targeting siRNA control measured byRT-qPCR. Data is presented as the mean±SD of a single experiment.

FIG. 22 shows Knockdown of PLG mRNA by Conjugate 21 throughreceptor-mediated uptake into primary human hepatocytes 24 hours postsiRNA treatment. PLG mRNA expression levels were normalised to ACTB andrelative to cells treated with a non-targeting siRNA control measured byRT-qPCR. Data is presented as the mean±SD of a single experiment.

Conjugate 21 achieves high levels of LPA mRNA inhibition withoutaffecting the levels of APOB and PLG mRNAs.

Example 14

To test the efficacy of conjugate 21 in vivo, male cynomolgus monkeyswere used to perform a pharmacodynamic study. Animals were grouped into4 groups, 3 animals per group. Before dosing, serum was prepared toestablish the baseline Lp(a) level for each animal. On day 1, conjugate21 was formulated in 0.9% saline and each animal received a single doseof conjugate 21 at doses of 0.1, 0.3, 1.0 and 3.0 mg/kg.

Serial samples were taken over 29 days and Lp(a) levels were measured byELISA (Mercodia, Catalogue No. 10-1106-01Lot: 27736; Uppsala, Sweden).All values were normalised to the baseline values for each individualanimal taken before dosing and presented as a percentage of the startinglevel (FIG. 23). Doses of 1.0 and 3.0 mg/kg showed marked reduction ofthe serum Lp(a) levels (69.4 and 77.3% after 29 days respectively). Thedose dependent effect results in an ED₅₀ dose of 0.56 mg/kg (FIG. 24).These data indicate that a significant and sustained reduction in serumLp(a) is achievable through a single dose of conjugate 21. Based on thisdata, dosing would be expected to be infrequent, not more often thanonce every two months.

STATEMENTS OF INVENTION

The following are statements of the invention:

-   1. A nucleic acid for inhibiting expression of LPA in a cell,    comprising at least one duplex region that comprises at least a    portion of a first strand and at least a portion of a second strand    that is at least partially complementary to the first strand,    wherein said first strand is at least partially complementary to at    least a portion of RNA transcribed from the LPA gene, wherein said    first strand comprises a nucleotide sequence selected from the    following sequences: SEQ ID NO: 9, 5, 1, 3, 7, 11, 13, 15, 17, 19,    21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43.-   2. The nucleic acid of statement 1, wherein said first strand    comprises a nucleotide sequence of SEQ ID NO: 9, and optionally    wherein said second strand comprises a nucleotide sequence of SEQ ID    NO: 10; or wherein said first strand comprises a nucleotide sequence    of SEQ ID NO: 5, and optionally wherein said second strand comprises    a nucleotide sequence of SEQ ID NO: 6.-   3. A nucleic acid for inhibiting expression of LPA in a cell,    comprising at least one duplex region that comprises at least a    portion of a first strand and at least a portion of a second strand    that is at least partially complementary to the first strand,    wherein said first strand is at least partially complementary to at    least a portion of RNA transcribed from the LPA gene, wherein said    first strand comprises a nucleotide sequence selected from the    following sequences: SEQ ID NO: 9 or 5.-   4. The nucleic acid of any of the preceding statements, wherein said    first strand and/or said second strand are each from 17-35    nucleotides in length.-   5. The nucleic acid of any of the preceding statements, wherein the    at least one duplex region consists of 17-25, preferably 19-25,    consecutive nucleotide base pairs.-   6. The nucleic acid of any of the preceding statements, wherein the    nucleic acid:    -   a) is blunt ended at both ends; or    -   b) has an overhang at one end and a blunt end at the other; or    -   c) has an overhang at both ends.-   7. The nucleic acid of any of the preceding statements, wherein one    or more nucleotides on the first and/or second strand are modified,    to form modified nucleotides.-   8. The nucleic acid of any of the preceding statements, wherein the    nucleic acid comprises a phosphorothioate linkage between the    terminal one, two or three 3′ nucleotides and/or 5′ nucleotides of    one or both ends of the first and/or the second strand.-   9. The nucleic acid of any of the preceding statements, wherein the    nucleic acid is conjugated to a ligand.-   10. The nucleic acid of statement 9, wherein the ligand    comprises (i) one or more N-acetyl galactosamine (GalNAc) moieties    or derivatives thereof, and (ii) a linker, wherein the linker    conjugates the at least one GalNAc moiety or derivative thereof to    the nucleic acid.-   11. The nucleic acid of any of statements 9-10, wherein the nucleic    acid is conjugated to a ligand comprising a compound of formula (I):    [S—X¹—P—X²]₃-A-X³—  (I)    -   wherein:        -   S represents a saccharide, preferably wherein the saccharide            is N-acetyl galactosamine;        -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂—            wherein m is 1, 2, or 3;        -   P is a phosphate or modified phosphate, preferably a            thiophosphate;        -   X² is alkylene or an alkylene ether of the formula            (—CH₂)_(n)—O—CH₂— where n=1-6;        -   A is a branching unit;        -   X³ represents a bridging unit;        -   wherein a nucleic acid as defined in any of statements 1 to            8 is conjugated to        -   X³ via a phosphate or modified phosphate, preferably a            thiophosphate.-   12. The nucleic acid of any of statements 9-10, wherein the first    RNA strand is a compound of formula (X):

-   -   wherein b is 0 or 1; and    -   the second RNA strand is a compound of formula (XI):

-   -    wherein:        -   c and d are independently 0 or 1;        -   Z₁ and Z₂ are the RNA portions of the first and second RNA            strands respectively;        -   Y is O or S;        -   n is 0, 1, 2 or 3; and        -   L₁ is a linker to which a ligand is attached; and    -   wherein b+c+d is 2 or 3.

-   13. A nucleic acid for inhibiting expression of LPA in a cell,    comprising at least one duplex region that comprises at least a    portion of a first strand and at least a portion of a second strand    that is at least partially complementary to the first strand,    wherein said first strand is at least partially complementary to at    least a portion of RNA transcribed from the LPA gene, wherein said    first strand comprises, and preferably consists of, the nucleotide    sequence of SEQ ID NO: 9 and optionally, wherein said second strand    comprises, and preferably consists of, the nucleotide sequence of    SEQ ID NO. 10.

-   14. The nucleic acid of statement 13, wherein the nucleic acid is    conjugated to a ligand and has the following structure

-   -   wherein Z is a nucleic acid according of statement 13 and is        preferably conjugated to the 5′ end of the second strand.

-   15. The nucleic acid of statement 14, wherein the nucleic acid    comprises two phosphorothioate linkages between each of the three    terminal 3′ and between each of the three terminal 5′ nucleotides on    the first strand, and two phosphorothioate linkages between the    three terminal nucleotides of the 3′ end of the second strand and    wherein the ligand is conjugated to the 5′ end of the second strand.

-   16. The nucleic acid of statement 13, wherein the nucleic acid is    conjugated to a ligand, wherein the first RNA strand is a compound    of formula (XV):

-   -   wherein b is 0 or 1; and    -   the second RNA strand is a compound of formula (XVI):

-   -   wherein c and d are independently 0 or 1;    -   wherein:    -   Z₁ and Z₂ are the RNA portions of the first and second RNA        strands respectively;    -   Y is O or S;    -   R₁ is H or methyl;    -   n is 0, 1, 2 or 3; and    -   L is the same or different in formulae (XV) and (XVI) and is        selected from the group consisting of:        -   —(CH₂)_(q), wherein q=2-12;        -   —(CH₂)_(r)—C(O)—, wherein r=2-12;        -   —(CH₂—CH₂—O)_(s)—CH₂—C(O)—, wherein s=1-5;        -   —(CH₂)_(t)—CO—NH—(CH₂)_(t)—NH—C(O)—, wherein t is            independently is 1-5;        -   —(CH₂)_(u)—CO—NH—(CH₂)_(u)—C(O)—, wherein u is independently            is 1-5; and        -   —(CH₂)_(v)—NH—C(O)—, wherein v is 2-12; and    -   wherein the terminal C(O), if present, is attached to the NH        group;    -   and wherein b+c+d is 2 or 3.

-   17. The nucleic acid of any of statements 13-16, comprising a    sequence and modifications as shown below:

SEQ ID NO: sequence modifications 9 5′ AUAACUCUGUCCAUUACCG 36162717181736152738 10 5′ CGGUAAUGGACAGAGUUAU 3′ 3845261846364645161

-   -   wherein, the specific modifications are depicted by numbers    -   1=2F-dU,    -   2=2′F-dA,    -   3=2F-dC,    -   4=2F-dG,    -   5=2′-OMe-rU;    -   6=2′-OMe-rA;    -   7=2′-OMe-rC;    -   8=2′-OMe-rG.

-   18. The nucleic acid of any of statements 13-16, wherein the    nucleotides at positions 2 and 14 from the 5′ end of the first    strand are modified with a 2′ fluoro modification, and the    nucleotides on the second strand which correspond to position 11, or    13, or 11 and 13, or 11-13 of the first strand are modified with a    2′ fluoro modification.

-   19. A composition comprising a nucleic acid of any of statements    1-18 and optionally a delivery vehicle and/or a physiologically    acceptable excipient and/or a carrier and/or a diluent and/or a    buffer and/or a preservative, for use as a medicament, preferably    for the prevention or treatment or risk reduction of a disease or    pathology, wherein the disease or pathology preferably is a    cardiovascular disease, wherein the cardiovascular disease    preferably is a stroke, atherosclerosis, thrombosis, a coronary    heart disease or aortic stenosis and/or any other disease or    pathology associated to elevated levels of Lp(a)-containing    particles.

-   20. A pharmaceutical composition comprising a nucleic acid of any of    statements 1-18 and further comprising a delivery vehicle,    preferably liposomes and/or a physiologically acceptable excipient    and/or a carrier and/or a diluent.

-   21. Use of a nucleic acid of any of statements 1-18 or a    pharmaceutical composition of statement 20 for the prevention or    treatment or risk reduction of a disease or pathology, wherein the    disease or pathology preferably is a cardiovascular disease, wherein    the cardiovascular disease preferably is a stroke, atherosclerosis,    thrombosis, a coronary heart disease or aortic stenosis and any    other disease or pathology associated to elevated levels of    Lp(a)-containing particles.

-   22. A method of preventing or treating a disease, disorder or    syndrome comprising administering a composition comprising a nucleic    acid of any of statements 1-18 or a composition according to    statements 19-20 to an individual in need of treatment, preferably    wherein the nucleic acid or composition is administered to the    subject subcutaneously, intravenously or using any other application    routes such as oral, rectal or intraperitoneal.

Summary sequence table SEQ ID Unmodified Sequence NO NameSequence (5′-3′) counterpart (5′-3′) 1 LPA-1014 first strandUCGUAUAACAAUAAGGGGC UCGUAUAACAAUAAGGGGC 2 LPA-1014 second strandGCCCCUUAUUGUUAUACGA GCCCCUUAUUGUUAUACGA 3 LPA-1024 first strandGAUAACUCUGUCCAUUACC GAUAACUCUGUCCAUUACC 4 LPA-1024 second strandGGUAAUGGACAGAGUUAUC GGUAAUGGACAGAGUUAUC 5 LPA-1038 first strandAUAACUCUGUCCAUUACCA AUAACUCUGUCCAUUACCA 6 LPA-1038 second strandUGGUAAUGGACAGAGUUAU UGGUAAUGGACAGAGUUAU 7 LPA-1040 first strandUAACUCUGUCCAUUACCGU UAACUCUGUCCAUUACCGU 8 LPA-1040 second strandACGGUAAUGGACAGAGUUA ACGGUAAUGGACAGAGUUA 9 LPA-1041 first strandAUAACUCUGUCCAUUACCG AUAACUCUGUCCAUUACCG 10 LPA-1041 second strandCGGUAAUGGACAGAGUUAU CGGUAAUGGACAGAGUUAU 11 LPA-1055 first strandAGAAUGUGCCUCGAUAACU AGAAUGUGCCUCGAUAACU 12 LPA-1055 second strandAGUUAUCGAGGCACAUUCU AGUUAUCGAGGCACAUUCU 13 LPA-1057 first strandAUAACUCUGUCCAUCACCA AUAACUCUGUCCAUCACCA 14 LPA-1057 second strandUGGUGAUGGACAGAGUUAU UGGUGAUGGACAGAGUUAU 15 LPA-1058 first strandAUAACUCUGUCCAUCACCU AUAACUCUGUCCAUCACCU 16 LPA-1058 second strandAGGUGAUGGACAGAGUUAU AGGUGAUGGACAGAGUUAU 17 LPA-1061 first strandUAACUCUGUCCAUUACCAU UAACUCUGUCCAUUACCAU 18 LPA-1061 second strandAUGGUAAUGGACAGAGUUA AUGGUAAUGGACAGAGUUA 19 LPA-1086 first strandAUGUGCCUUGAUAACUCUG AUGUGCCUUGAUAACUCUG 20 LPA-1086 second strandCAGAGUUAUCAAGGCACAU CAGAGUUAUCAAGGCACAU 21 LPA-1099 first strandAGUUGGUGCUGCUUCAGAA AGUUGGUGCUGCUUCAGAA 22 LPA-1099 second strandUUCUGAAGCAGCACCAACU UUCUGAAGCAGCACCAACU 23 LPA-1102 first strandAAUAAGGGGCUGCCACAGG AAUAAGGGGCUGCCACAGG 24 LPA-1102 second strandCCUGUGGCAGCCCCUUAUU CCUGUGGCAGCCCCUUAUU 25 LPA-1116 first strandUAACUCUGUCCAUCACCAU UAACUCUGUCCAUCACCAU 26 LPA-1116 second strandAUGGUGAUGGACAGAGUUA AUGGUGAUGGACAGAGUUA 27 LPA-1127 first strandAUGAGCCUCGAUAACUCUG AUGAGCCUCGAUAACUCUG 28 LPA-1127 second strandCAGAGUUAUCGAGGCUCAU CAGAGUUAUCGAGGCUCAU 29 LPA-1128 first strandAAUGAGCCUCGAUAACUCU AAUGAGCCUCGAUAACUCU 30 LPA-1128 second strandAGAGUUAUCGAGGCUCAUU AGAGUUAUCGAGGCUCAUU 31 LPA-1141 first strandAAUGCUUCCAGGACAUUUC AAUGCUUCCAGGACAUUUC 32 LPA-1141 second strandGAAAUGUCCUGGAAGCAUU GAAAUGUCCUGGAAGCAUU 33 LPA-1151 first strandACAGUGGUGGAGAAUGUGC ACAGUGGUGGAGAAUGUGC 34 LPA-1151 second strandGCACAUUCUCCACCACUGU GCACAUUCUCCACCACUGU 35 LPA-1171 first strandGUAUGUGCCUCGAUAACUC GUAUGUGCCUCGAUAACUC 36 LPA-1171 second strandGAGUUAUCGAGGCACAUAC GAGUUAUCGAGGCACAUAC 37 LPA-1177 first strandUCGAUAACUCUGUCCAUCA UCGAUAACUCUGUCCAUCA 38 LPA-1177 second strandUGAUGGACAGAGUUAUCGA UGAUGGACAGAGUUAUCGA 39 LPA-1189 first strandUGUCACUGGACAUUGUGUC UGUCACUGGACAUUGUGUC 40 LPA-1189 second strandGACACAAUGUCCAGUGACA GACACAAUGUCCAGUGACA 41 LPA-1244 first strandCUGGGAUCCAUGGUGUAAC CUGGGAUCCAUGGUGUAAC 42 LPA-1244 second strandGUUACACCAUGGAUCCCAG GUUACACCAUGGAUCCCAG 43 LPA-1248 first strandAGAUGACCAAGCUUGGCAG AGAUGACCAAGCUUGGCAG 44 LPA-1248 second strandCUGCCAAGCUUGGUCAUCU CUGCCAAGCUUGGUCAUCU 45 LPA: (upper) humanAAGTGTCCTTGCGACGTCC AAGTGTCCTTGCGACGTCC 46 LPA: (lower) humanCCTGGACTGTGGGGCTTT CCTGGACTGTGGGGCTTT 47 LPA: (probe) humanCTGTTTCTGAACAAGCACCAACGGAGC CTGTTTCTGAACAAGCACCAACGG GC 48LPA (upper) cynomolgus GTGTCCTCGCAACGTCCA GTGTCCTCGCAACGTCCA 49LPA (lower) cynomolgus GACCCCGGGGCTTTG GACCCCGGGGCTTTG 50LPA (probe) cynomolgus TGGCTGTTTCTGAACAAGCACCAATGGTGGCTGTTTCTGAACAAGCACCAA TGG 51 APOB (upper) humanTCATTCCTTCCCCAAAGAGACC TCATTCCTTCCCCAAAGAGACC 52 APOB (lower) humanCACCTCCGTTTTGGTGGTAGAG CACCTCCGTTTTGGTGGTAGAG 53 APOB (probe) humanCAAGCTGCTCAGTGGAGGCAACACATTA CAAGCTGCTCAGTGGAGGCAACAC ATTA 54beta-Actin (upper)  GCATGGGTCAGAAGGATTCCTAT GCATGGGTCAGAAGGATTCCTAThuman 55 beta-Actin (lower)  TGTAGAAGGTGTGGTGCCAGATTTGTAGAAGGTGTGGTGCCAGATT human 56 beta-Actin (probe) TCGAGCACGGCATCGTCACCAA TCGAGCACGGCATCGTCACCAA human 57beta-Actin (upper) AAGGCCAACCGCAGAAG AAGGCCAACCGCGAGAAG cynomolgus 58beta-Actin AAGGCCAACCGCGAGAAG AGAGGCGTACAGGGACAGCA (lower) cynomolgus 59beta-Actin TGAGACCTTCAACACCCCGCCATGTAC TGAGACCTTCAACACCCCAGCCAT(probe) cynomolgus GTAC 60 PPIB (upper) human AGATGTAGGCCGGGTGATCTTTAGATGTAGGCCGGGTGATCTTT 61 PPIB (lower) human GTAGCCAAATCCTTTCTCTCCTGTGTAGCCAAATCCTTTCTCTCCTGT 62 PPIB (probe) humanTGTTCCAAAAACAGTGGATAATTTTGTGGCC TGTTCCAAAAACAGTGGATAATTT TGTGGCC 63LPA: (upper) human AAGTGTCCTTGCGACGTCC AAGTGTCCTTGCGACGTCC 64LPA: (lower) human CCTGGACTGTGGGGCTTT CCTGGACTGTGGGGCTTT 65LPA: (probe) human CTGTTTCTGAACAAGCACCAACGGAGC CTGTTTCTGAACAAGCACCAACGGAGC 66 LPA (upper) cynomolgus GTGTCCTCGCAACGTCCA GTGTCCTCGCAACGTCCA 67LPA (lower) cynomolgus GACCCCGGGGCTTTG GACCCCGGGGCTTTG 68LPA (probe) cynomolgus TGGCTGTTTCTGAACAAGCACCAATGGTGGCTGTTTCTGAACAAGCACCAT GG 69 APOB (upper) human TCATTCCTTCCCCAAAGAGACCTCATTCCTTCCCCAAAGAGACC 70 APOB (lower) human CACCTCCGTTTTGGTGGTAGAGCACCTCCGTTTTGGTGGTAGAG 71 APOB (probe) humanCAAGCTGCTCAGTGGAGGCAACACATTA CAAGCTGCTCAGTGGAGGCAACAC ATTA 72beta-Actin (upper)  GCATGGGTCAGAAGGATTCCTAT GCATGGGTCAGAAGGATTCCTAThuman 73 beta-Actin (lower)  TGTAGAAGGTGTGGTGCCAGATTTGTAGAAGGTGTGGTGCCAGATT human 74 beta-Actin (probe) TCGAGCACGGCATCGTCACCAA TCGAGCACGGCATCGTCACCAA human 75Modified SEQ ID NO: 1 5381616272616284847 UCGUAUAACAAUAAGGGGC 76Modified SEQ ID NO: 2 4737351615451616382 GCCCCUUAUUGUUAUACGA 77Modified SEQ ID NO: 3 8252635354537251637 GAUAACUCUGUCCAUUACC 78Modified SEQ ID NO: 4 4816254827282815253 GGUAAUGGACAGAGUUAUC 79Modified SEQ ID NO: 5 6162717181736152736 AUAACUCUGUCCAUUACCA 80Modified SEQ ID NO: 6 1845261846364645161 UGGUAAUGGACAGAGUUAU 81Modified SEQ ID NO: 7 5263535453725163745 UAACUCUGUCCAUUACCGU 82Modified SEQ ID NO: 8 2748162548272828152 ACGGUAAUGGACAGAGUUA 83Modified SEQ ID NO: 9 6162717181736152738 AUAACUCUGUCCAUUACCG 84Modified SEQ ID NO: 10 3845261846364645161 CGGUAAUGGACAGAGUUAU 85Modified SEQ ID NO: 11 6462545473538252635 AGAAUGUGCCUCGAUAACU 86Modified SEQ ID NO: 12 2815253828472725171 AGUUAUCGAGGCACAUUCU 87Modified SEQ ID NO: 13 6162717181736172736 AUAACUCUGUCCAUCACCA 88Modified SEQ ID NO: 14 1845461846364645161 UGGUGAUGGACAGAGUUAU 89Modified SEQ ID NO: 15 6162717181736172735 AUAACUCUGUCCAUCACCU 90Modified SEQ ID NO: 16 2845461846364645161 AGGUGAUGGACAGAGUUAU 91Modified SEQ ID NO: 17 5263535453725163725 UAACUCUGUCCAUUACCAU 92Modified SEQ ID NO: 18 2548162548272828152 AUGGUAAUGGACAGAGUUA 93Modified SEQ ID NO: 19 6181837154616271718 AUGUGCCUUGAUAACUCUG 94Modified SEQ ID NO: 20 3646451617264836361 CAGAGUUAUCAAGGCACAU 95Modified SEQ ID NO: 21 6451845471835172826 AGUUGGUGCUGCUUCAGAA 96Modified SEQ ID NO: 22 1535462836472736271 UUCUGAAGCAGCACCAACU 97Modified SEQ ID NO: 23 6252648483547363648 AAUAAGGGGCUGCCACAGG 98Modified SEQ ID NO: 24 3718184728373715251 CCUGUGGCAGCCCCUUAUU 99Modified SEQ ID NO: 25 5263535453725363725 UAACUCUGUCCAUCACCAU 100Modified SEQ ID NO: 26 2548182548272828152 AUGGUGAUGGACAGAGUUA 101Modified SEQ ID NO: 27 6182837174616271718 AUGAGCCUCGAUAACUCUG 102Modified SEQ ID NO: 28 3646451617464835361 CAGAGUUAUCGAGGCUCAU 103Modified SEQ ID NO: 29 6254647353825263535 AAUGAGCCUCGAUAACUCU 104Modified SEQ ID NO: 30 2828152538284717251 AGAGUUAUCGAGGCUCAUU 105Modified SEQ ID NO: 31 6254715372846361517 AAUGCUUCCAGGACAUUUC 106Modified SEQ ID NO: 32 4626181735482647251 GAAAUGUCCUGGAAGCAUU 107Modified SEQ ID NO: 33 6364548184646254547 ACAGUGGUGGAGAAUGUGC 108Modified SEQ ID NO: 34 4727251717363727181 GCACAUUCUCCACCACUGU 109Modified SEQ ID NO: 35 8161818371746162717 GUAUGUGCCUCGAUAACUC 110Modified SEQ ID NO: 36 4645161746483636163 GAGUUAUCGAGGCACAUAC 111Modified SEQ ID NO: 37 5382526353545372536 UCGAUAACUCUGUCCAUCA 112Modified SEQ ID NO: 38 1825482728281525382 UGAUGGACAGAGUUAUCGA 113Modified SEQ ID NO: 39 5453635482725181817 UGUCACUGGACAUUGUGUC 114Modified SEQ ID NO: 40 4636362545372818272 GACACAAUGUCCAGUGACA 115Modified SEQ ID NO: 41 7184825372548181627 CUGGGAUCCAUGGUGUAAC 116Modified SEQ ID NO: 42 4516363725482537364 GUUACACCAUGGAUCCCAG 117Modified SEQ ID NO: 43 6461827362835184728 AGAUGACCAAGCUUGGCAG 118Modified SEQ ID NO: 44 3547362835184536171 CUGCCAAGCUUGGUCAUCU 119GalNac-LPA-1038-L1 OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-AUAACUCUGUCCAUUACCA first strand FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeA 120 GalNac-LPA-1038-L1[ST23 (ps)]3 long trebler (ps)FU-OMeG-FG-OMeU- UGGUAAUGGACAGAGUUAUsecond strand FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 121 GalNac-LPA-1038-L6OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC- AUAACUCUGUCCAUUACCA first strandFU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA- OMeC-(ps)-FC-(ps)-OMeA 122GalNac-LPA-1038-L6 [ST23 (ps)]3 ST43 (ps)FU-OMeG-FG-OMeU-FA-UGGUAAUGGACAGAGUUAU second strand OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 123 GalNac-LPA-1041-L1 OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC- AUAACUCUGUCCAUUACCG first strandFU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA- OMeC-(ps)-FC-(ps)-OMeG 124GalNac-LPA-1041-L1 [ST23 (ps)]3 long trebler (ps) FC-OMeG-FG-OMeU-CGGUAAUGGACAGAGUUAU second strand FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 125 GalNac-LPA-1041-L6 OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC- AUAACUCUGUCCAUUACCG first strandFU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA- OMeC-(ps)-FC-(ps)-OMeG 126GalNac-LPA-1041-L6 [ST23 (ps)]3 ST43 (ps) FC-OMeG-FG-OMeU-FA-CGGUAAUGGACAGAGUUAU second strand OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 127 STS16001AL33mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG UUAUAGAGCAAGAACACUGUUmA fA mC fA mC fU mG (ps) fU (ps) mU (ps) Ser(GN) 128 STS16001BL20Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU AACAGUGUUCUUGCUCUAUAAmU fG mC fU mC fU mA fU (ps) mA (ps) fA 129 STS16001AmU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG UUAUAGAGCAAGAACACUGUUmA fA mC fA mC fU mG (ps) fU (ps) mU 130 STS16001BV1L42Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU AACAGUGUUCUUGCUCUAUAAfU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) Ser(GN) 131STS16001V1B fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mUAACAGUGUUCUUGCUCUAUAA fG mC fU mC fU mA fU (ps) mA (ps) fA 132 STS18001AmU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC UCGAAGUAUUCCGCGUACGmG fC mG fU mA (ps) fC (ps) mG 133 STS18001BL4[(ST23) (ps)]₃ C4XLT (ps) fC mG fU mA fC mG fC CGUACGCGGAAUACUUCGAmG fG mA fA mU fA mC fU mU fC (ps) mG (ps) fA 134 STS16001BL4[(ST23) (ps)]3 C4XLT(ps) fA (ps) mA (ps) fC mA fG AACAGUGUUCUUGCUCUAUAAmU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA 135 X0373AmA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC AUAACUCUGUCCAUUACCGmA fU mU fA mC (ps) fC (ps) mG 136 X0373BSer(GN) (ps) fC (ps) mG (ps) fG mU fA mA fU mG CGGUAAUGGACAGAGUUAUfG mA fC mA fG mA fG mU fU (ps) mA (ps) fU (ps) Ser(GN) 137 STS2041BST23 (ps) ST23 (ps) ST23 (ps) C6XLT (ps) fC mG CGGUAAUGGACAGAGUUAUfG mU fA mA fU mG fG mA fC mA fG mA fG mU fU (ps) mA (ps) fU 138 X0125AmC (ps) fU (ps) mU fA mC fU mC fU mC fG mC fC CUUACUCUCGCCCAAGCGAmC fA mA fG mC (ps) fG (ps) mA 139 X0125B[(ST23) (ps)]₃ (C6XLT) (ps) fU mC fG mC fU mU fG UCGCUUGGGCGAGAGUAAGmG fG mC fG mA fG mA fG mU fA (ps) mA (ps) fG 140 Probe based on SEQ BHQ1-TGGCTGTTTCTGAACAAGCACCATGG- TGGCTGTTTCTGAACAAGCACCAA ID NO: 50 FAMTGG 141 Probe based on SEQ  BHQ1-TCGAGCACGGCATCGTCACCAA-VICTCGAGCACGGCATCGTCACCAA ID NO: 56 142 STS16001BV1L75Ser(GN) fA (ps) mA (ps) fC mA fG mU fG mU fU AACAGUGUUCUUGCUCUAUAAmC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA Ser(GN) 143STS16001BV16L42 Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fUAACAGUGUUCUUGCUCUAUAA mU fG mC fU mC fU mA fU mA fA (ps) Ser(GN) 144STS16001BV20L75 Ser(GN) fA mA fC mA fG mU fG mU fU mC fU mUAACAGUGUUCUUGCUCUAUAA fG mC fU mC fU mA fU mA fA Ser(GN) 145STS16001BV1L94 Ser(GN) (ps) Ser(GN) (ps) fA (ps) mA (ps) fC mA fGAACAGUGUUCUUGCUCUAUAA mU fG mU fU mC fU mU fG mC fU mC fU mA fU(ps) mA (ps) fA (ps) Ser(GN) (ps) Ser(GN) 146 STS16001V1BL96C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG AACAGUGUUCUUGCUCUAUAAmU fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) C7Am(GN) 147STS16001V1BL97 GlyC3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fGAACAGUGUUCUUGCUCUAUAA mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA(ps) fA (ps) GlyC3Am(GN) 148 Conjugate 10 secondPipAm(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG AACAGUGUUCUUGCUCUAUAAstrand mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA(ps) fA (ps) PipAm(GN) 149 STS16001V1BL88C3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG AACAGUGUUCUUGCUCUAUAAmU fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) C3Am(GN) 150STS16001V1BL87 C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mUAACAGUGUUCUUGCUCUAUAA fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA(ps) GlyC3Am(GN) 151 Conjugate 15 antisensemU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU UCUUCUUAAACUGAGUUUC strandmG fA mG fU mU (ps) fU (ps) mC 152 Conjugate 15 senseSer(GN) (ps) fG (ps) mA (ps) fA mA fC mU fC mA GAAACUCAGUUUAAGAAGAstrand fG mU fU mU fA mA fG mA fA (ps) mG (ps) fA (ps) Ser(GN) 153Conjugate 16 antisense mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fGAUGUAGCCGAGGAUCUUCU strand mA fU mC fU mU (ps) fC (ps) mU 154Conjugate 16 antisense Ser(GN) (ps) fA (ps) mG (ps) fA mA fG mA fU mCAGAAGAUCCUCGGCUACAU strandfC mU fC mG fG mC fU mA fC (ps) mA (ps) fU (ps) Ser(GN) 155Conjugate 18 antisense mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fGAACCAGAAGAAGCAGGUGA strand mC fA mG fG mU (ps) fG (ps) mA 156Conjugate 18 sense Ser(GN) (ps) fU (ps) mC (ps) fA mC fC mU fG mCUCACCUGCUUCUUCUGGUU strandfU mU fC mU fU mC fU mG fG (ps) mU (ps) fU (ps) Ser(GN) 157 STS16001BV1fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU AACAGUGUUCUUGCUCUAUAAfG mC fU mC fU mA fU (ps) mA (ps) fA 158 Reference Conjugate 6[ST23 (ps)]3 ltrb (ps) fG mA fA mA fC mU fC mA fG GAAACUCAGUUUAAGAAGAsense strand mU fU mU fA mA fG mA fA (ps) mG (ps) fA 159Reference Conjugate 7 [ST23 (ps)]3 ltrb (ps) fA mG fA mA fG mA fU mC fCAGAAGAUCCUCGGCUACAU sense strand mU fC mG fG mC fU mA fC (ps) mA (ps) fU160 Reference Conjugate 8 mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fGUACCAGAAGAAGCAGGUGA antisense strand mC fA mG fG mU (ps) fG (ps) mA 161Reference Conjugate 8 [ST23 (ps)]3 ST41 (ps)fU mC fA mC fC mU fG mCUCACCUGCUUCUUCUGGUA sense strandfU mU fC mU fU mC fU mG fG (ps) mU (ps) fA 162 Reference Conjugate 9[ST23 (ps)]3 C6XLT (ps) fC mG fG mU fA mA fU CGGUAAUGGACAGAGUUAUsense strand mG fG mA fC mA fG mA fG mU fU (ps) mA (ps) fU 163Conjugate 21 sense mC mG mG mU mA mA fU fG fG mA mC mA mGCGGUAAUGGACAGAGUUAU strand without ligand mA mG mU mU (ps) mA (ps) mU164 Conjugate 21 sense [ST23 (ps)]3 C6XLT (ps) mC mG mG mU mA mA fUCGUAAUGGACAGAGUUAU strand fG fG mA mC mA mG mA mG mU mU (ps) mA (ps) mU165 Conjugate 21 antisense mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fCAUAACUCUGUCCAUUACCG strand mA fU mU fA mC (ps) fC (ps) mG

A single sequence may have more than one name. In those cases, one ofthose names is given in the summary sequence table.

Where specific linkers and or modified linkages are taught within an RNAsequence, such as (ps) and [ST23 (ps)]3 ST41 (ps) etc, these areoptional parts of the sequence, but are a preferred embodiment of thatsequence.

The following abbreviations may be used, particularly in listedsequences:

Abbreviation Meaning 1 2′F-dU 2 2′F-dA 3 2′F-dC 4 2′F-dG 5 2′OMe-rU 62′OMe-rA 7 2′OMe-rC 8 2′OMe-rG mA, mU, mG, 2′deoxy-2′-F RNA OMeA, OMeU,OMeC, OMeG 2′-OMe 2′-O-Methyl modification fA, fU, fC, fG 2′ deoxy-2′-FRNA nucleotides 2′-F, 2′-fluoro, 2′ 2′-fluoro modification fluoro (ps)phosphorothioate (vp) Vinyl-(E)-phosphonate ivA, ivC, ivU, inverted RNA(3′-3′) ivG FAM 6-Carboxyfluorescein BHQ Black Hole Quencher 1 ST23

ST41/C4XLT

ST43 (or C6XLT)

ST43-phos/ C6XLT-phos

Long trebler /Itrb/STKS (phosphoramidite)

Ser(GN)

Ser(GN) (phosphoramidite)

C3Am(GN)

GlyC3Am(GN)

C6Am(GN)

C7Am(GN)

PipAm(GN)

[ST23 (ps)]3 C4XLT (ps) = [ST23 (ps)]3 ST41 (ps) = L4

[ST23 (ps)]3 C6XLT (ps) = [ST23 (ps)]3 ST43 (ps) = L6

The invention claimed is:
 1. A nucleic acid comprising at least oneduplex region that comprises at least a portion of a first strand and atleast a portion of a second strand that is at least partiallycomplementary to the first strand, wherein said first strand comprisesthe nucleic acid sequence of SEQ ID NO: 165 and the second strandcomprises the nucleic acid sequence of SEQ ID NO:
 163. 2. The nucleicacid of claim 1, wherein the first strand consists of SEQ ID NO:
 165. 3.The nucleic acid of claim 1, wherein the second strand consists of SEQID NO:
 163. 4. The nucleic acid of claim 1, wherein the first strandconsists of SEQ ID NO: 165 and the second strand consists of SEQ ID NO:163.
 5. The nucleic acid of claim 1, wherein the nucleic acid isconjugated to a ligand.
 6. The nucleic acid of claim 5, wherein theligand comprises (i) one or more N-acetyl galactosamine (GalNAc)moieties or derivatives thereof, and (ii) a linker, wherein the linkerconjugates the at least one GalNAc moiety or derivative thereof to thenucleic acid.
 7. The nucleic acid of claim 1, wherein the nucleic acidis conjugated to a ligand comprising a compound of formula (I):[S—X¹—P—X²]₃-A-X³—  (I) wherein: S represents a saccharide; X¹represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein m is 1, 2,or 3; P is a phosphate or modified phosphate; X² is alkylene or analkylene ether of the formula (—CH₂)_(n)—O—CH₂— where n=1-6; A is abranching unit; X³ represents a bridging unit; and wherein a nucleicacid as defined in claim 1 is conjugated to X³ via a phosphate ormodified phosphate.
 8. The nucleic acid of claim 1, wherein the nucleicacid is conjugated to a ligand and has the following structure:

wherein Z is a nucleic acid according to claim
 1. 9. The nucleic acid ofclaim 5, wherein the ligand is conjugated to the 5′ end of the secondstrand.
 10. The nucleic acid of claim 5, wherein the second strandconsists of SEQ ID NO:
 164. 11. A pharmaceutical composition comprisinga nucleic acid of claim 1 and further comprising a delivery vehicleand/or a physiologically acceptable excipient and/or a carrier and/or adiluent.
 12. A method of preventing or treating a cardiovascular diseaseor a disease associated with elevated levels of Lp(a) particles, themethod comprising administering a composition comprising a nucleic acidof claim 1 to an individual in need thereof.
 13. The method of claim 12,wherein the composition is administered subcutaneously or intravenously.14. The method of claim 12, wherein the disease is a cardiovasculardisease.
 15. The method of claim 14, wherein the cardiovascular diseaseis stroke, atherosclerosis, thrombosis, a coronary heart disease, oraortic stenosis.
 16. The method of claim 12, wherein the disease is adisease associated with elevated levels of Lp(a) particles.
 17. Themethod of claim 12, wherein the first strand consists of SEQ ID NO: 165.18. The method of claim 12, wherein second strand consists of SEQ ID NO:163.
 19. The method of claim 12, wherein the first strand consists ofSEQ ID NO: 165 and the second strand consists of SEQ ID NO:
 163. 20. Themethod of claim 12, wherein the nucleic acid is conjugated to a ligandand has the following structure:

wherein Z is a nucleic acid according to claim
 1. 21. The method ofclaim 14, wherein the nucleic acid is conjugated to a ligand and has thefollowing structure:

wherein Z is a nucleic acid according to claim
 1. 22. The method ofclaim 15, wherein the nucleic acid is conjugated to a ligand and has thefollowing structure:

wherein Z is a nucleic acid according to claim
 1. 23. The method ofclaim 16, wherein the nucleic acid is conjugated to a ligand and has thefollowing structure:

wherein Z is a nucleic acid according to claim
 1. 24. The method ofclaim 12, wherein the second strand consists of SEQ ID NO:
 164. 25. Themethod of claim 17, wherein the second strand consists of SEQ ID NO:164.
 26. The nucleic acid of claim 7, wherein S represents N-acetylgalactosamine; P is a thiophosphate; and wherein the nucleic acid asdefined in claim 1 is conjugated to X³ via a thiophosphate, and thenucleic acid is conjugated to X³ via the 5′ end of the second strand.27. The nucleic acid of claim 8, wherein the ligand is conjugated to the5′ end of the second strand.
 28. The nucleic acid of claim 8, whereinthe second strand consists of SEQ ID NO: 164.